The Mutagenesis Protocol

by Emooly (Henry M. Gunn High School) on 2016-02-22 14:06:12 PDT
Emily Cao
June 2015
Cui Lab
Several months ago, I had the opportunity to work in the Cui Lab.

This is a protocol that contains a lot of the technical skills I have learned.

Screen Shot 2016-02-22 at 2.05.07 PM
Frog oocytes -- these are the little guys I injected the RNA products into the membranes of and utilized electrophysiology in order to analyze fluctuations within their ion channel activities!

Protocol: Designing DNA Primers & PCR Products with the Utilization of Serial Cloner
 

Objective: The purpose of designing DNA primers and PCR products is so that the scientist is aware of the lengths of their PCR products in addition the sequence of their primers while working with the mutation DNA. These components will also provide the scientist with a general idea of the primers’ melting temperature as well as the locations in base pairs of their products.
 

Materials:
-Computer
-Serial Cloner Application
-Microsoft Word
-Access to the Cui Lab Cisco AnyConnect Network
-Printed Version of Template DNA Sequence Map
 

Procedure:
 
  1. Cytosine (C) and Guanine (G) have higher melting temperatures than Adenine (A) and Thymine (T). Therefore, C and G will be 4 degrees Celsius respectively and A and T will be 2 degrees Celsius respectively. Your target melting temperature is approximately 50 degrees Celsius, and you must end at either a G or a C due to the fact that they are more stable than A and T. Anything between 48 and 54 degrees Celsius is acceptable-- however anything less than or more should be reconsidered.
  2. In order to determine the melting temperature of Primer B, count directly left to the first mutated nucleotide. Stop at either a G or a C at approximately 50 degrees Celsius. Record the base pair in which the nucleotide you end at is located. Due to the fact that Primer B is antiparallel and moves to the left direction, this is where it technically ends.
  3. In order to determine the melting temperature of Primer C, count directly right to the last mutated nucleotide. Stop at either a G or a C at approximately 50 degrees Celsius. Record the base pair in which the nucleotide you end at is located. Due to the fact that Primer C is parallel and moves to the right direction, this is where it technically ends.
  4. Now you must determine the overlap points. In order to determine the beginning overlapping point, start at the first G or C to the right of Primer B’s endpoint and begin counting until you reach approximately 50 degrees Celsius. Record the position of the leftmost overlapping point as well as the position of the rightmost overlapping point.
  5. The distance between the rightmost point of the overlap and the endpoint of Primer B will be the total length of Primer B. Copy and paste this section into a new window of Serial Cloner, and click “Sequence” and then select “Antiparallel”. This will position Primer B appropriately due to the fact that it runs in an opposite direction along the DNA sequence. Title it appropriately, with the name of the mutation, its inventory number, as well as the proper letter of the primer.
  6. The distance between the leftmost point of the overlap and the endpoint of Primer C will be the total length of Primer C. Copy and paste this section into a new window of Serial Cloner. Title it appropriately, with the name of the mutation, its inventory number, as well as the proper letter of the primer.
  7. Now you can fill out the “2. Primers” section on your design. Make sure to write down the sequence of Primer B and Primer C, the length of them in mers, their melting temperatures, as well as the melting temperature of the overlap.
  8. This is the point in which you can start creating your PCR products. PCR product AB will be located from 220 to the rightmost overlap point. Copy and paste this section into a new window of Serial Cloner. Title it appropriately, with the name of the mutation, its inventory number, as well as the proper lettering of the PCR product.
  9. PCR product CD will be located from the leftmost overlap point to 1289. Copy and paste this section into a new window of Serial Cloner. Title it appropriately, with the name  of the mutation, its inventory number, as well as the proper lettering of the PCR product.
  10. PCR product AD will be located from 220 to 1289. Its length should be 1070. Copy and paste this section into a new window of Serial Cloner. Title it appropriately, with the name of the mutation, its inventory number, as well as the proper lettering of the PCR product.
  11. Now you can fill out the “3. PCR Products” section on your design. Make sure to write down the location of each PCR product. For AB, it will be 220-Rightmost Overlap Point in bps. For CD, it will be Leftmost Overlap Point-1289. For AD, it will be 220-1289. Record the length of these PCR products in base pairs by subtracting the smaller base pair value from the larger base pair value and adding one.
  12. Copy and paste the entire sequence of the mutation DNA, which should have a length of 7852 bps into a new window. Title it appropriately, with the name of the mutation in addition to its inventory number.
  13. Store all of your files into a folder. Title it appropriately, with the name of the mutation in addition to its inventory number. The folder should contain:
    1. Microsoft Word DNA Mutation Design
    2. Serial Cloner Primer B Sequence (Double check to see that it is antiparallel)
    3. Serial Cloner Primer C Sequence  
    4. Serial Cloner PCR Product AB Sequence
    5. Serial Cloner PCR Product CD Sequence
    6. Serial Cloner PCR Product AD Sequence
    7. Serial Cloner Mutation DNA Sequence
  14. All of your lengths should match up with one another, and all of the information within your Serial Cloner files should be recorded in the DNA Mutation Design on Microsoft Word.
  15. Update the mutation progress spreadsheet on the Cui Lab’s Cisco AnyConnect network. After all of your work has been checked and cleared, you can place the order of your primers from the Integrated DNA Technology company.
 

Protocol: Designing a Point Mutation in DNA with the Utilization of Serial Cloner
 

Objective: The purpose of designing a point mutation with the utilization of the Serial Cloner application is so that the scientist is clear about exactly what to do throughout the experiment. When a mutation is designed, the scientist will be aware of exactly what type of mutation they are working with, the type of restriction enzyme they are supposed to use, and a general idea of what the mutation DNA will look like compared to the wild type DNA after gel electrophoresis.
 

Materials:
-Computer
-Serial Cloner Application
-Microsoft Word
-Access to the Cui Lab Cisco AnyConnect Network
-Printed Version of Template DNA Sequence Map
 

Procedure:
 
  1. Open a sequence of a DNA template in order to start your mutation. For example, the file for the wild type of the Kv ion channel is titled “KvLQT1-WT-pcDNA3.1”.
  2. Click on the “Sequence Map” button. This will show exactly where all of your codons and the correlating amino acids are.
  3. Assign an inventory number to your specific mutation. In this case, “E261N” will be associated with “(Kv708)”. This should be on the mutation progress sheet. “E” represents the original amino acid, and “N” signifies the desired amino acid.
  4. Multiply the number in between the amino acids by 3. For instance, 261 X 3 = 783-- this is the amount in base pairs  where the amino acid you want to change will be.
  5. Start by opening a design template, and begin to fill in the “1. Mutations” section. Now that you know 783 bps is location of your amino acid, identify the sequence of its codons. Once you know the sequence of its codons, make sure to write down that its original place is “781-gag-783”.
  6. Refer to the universal code chart, and start by making “silent mutations”. This is when a nucleotide is changed, but the amino acid that is produced will not be interfered with. Begin a systematic process by testing all of the different nucleotide combinations. Make sure to record your nucleotide adjustments as well. For instance, “781-gag-783 ? 781-Aag-783” or “781-gag-783 ? 781-AaA-783”. Ensure that all of the nucleotide changes are capitalized so it is clear that they are mutated.
  7. Make the changes in the original window, and then open the sequence map. Record any changes in the restriction enzyme sites. For instance, “Lost AceIII restriction site”, or “Lost AluI restriction site” or “Lost AluBI restriction site.”
  8. Once you have written down the restriction enzyme sites that have been adjusted, check the latest version of the New England BioLabs catalog to see that your restriction enzyme is available.
  9. If your restriction enzyme is available in the catalog, make sure to check the conditions of the restriction enzyme. If it causes methylation, it will not work, and you will have to use a different restriction enzyme. Methylation is indicated by “dam” or “dcm”.
  10. Once you find a restriction enzyme that is available in the catalog and does not cause methylation, open up the graphic map of your wild type template DNA as well as the graphic map of your mutation DNA.
  11. Compare to see how many restriction sites your enzyme possesses. If there are way too many, your restriction enzyme will not work due to the fact that it will be difficult to track the change during MiniPrep. If there are very few, the restriction enzyme will work because the difference between the wild type DNA and the mutation DNA will be obvious.
  12. If after making numerous adjustments to your desired amino acid’s codon does not work, move upstream, and make a silent mutation. For example, “784-cag-786 ? 784-caA-786”.
  13. The more restriction enzyme sites that are added or removed, the more promising the mutation will be. Continue this process until you find a restriction enzyme that it is available in the New England BioLabs catalog, does not cause methylation, and has so few cute sites that it is noticeable enough to differentiate between the wild type DNA and the mutation DNA.
  14. Now you can fill out “The Mutant” section by indicating the restriction enzyme you desire to use in order to check for the presence of the mutation, the buffer and temperature in which it works at, as well as the bands in base pairs that you expect it to appear. You can also include a picture from the “Virtual Cutter” setting of Serial Cloner that displays a side by side comparison of the wild type DNA and the mutation DNA.
  15. Once all of your work has been completed, cleared, and checked, you can see if the laboratory has your restriction enzyme in its inventory. If not, you can place your order for a new restriction enzyme from the New England Biolabs company.
 

Protocol: Diluting DNA Primers to Original Condition and to Working Condition
 

Objective: In order to appropriately utilize the primers you have ordered from the Integrated DNA Technology company, you must dilute it so that it can be used properly. All of the materials utilized throughout the experiment must have a consistent concentration in order to be functional within the procedure.
 

Materials:
  • Integrated DNA Technology Primer B
  • Integrated DNA Technology Primer C
  • Ultra Pure Water
  • 200 uL Micropipet
  • 1.5 Microcentrifuge Tube
  • Laboratory Coat
  • Gloves
  • Facial Protection Gear, i.e. Goggles (Optional)
 

Procedure:
 
  1. Open the package from the Integrated DNA Technology. This envelope should contain your primers stored in small plastic containers as well as their order forms. Remove the stickers on the order forms of your primers and place them into your laboratory notebook. Identify the original concentration of your primers in nanomoles.
  2. Multiply the original concentration of your primers in nanomoles by 4. For instance: (Kv678 Primer B’s original concentration: 23.9 Nanomoles X 4 = 95.6 Lambda, Kv678 Primer C’s original concentration: 34.1 Nanomoles X 4 = 136.4 Lambda).
  3. This amount is the volume of ultra pure water you must transfer into the small plastic containers that store your primers. This process will dilute your primers into the concentration we want prior to working condition, which is 250 micromoles.
  4. Put on your personal protective equipment, which should include your laboratory coat, gloves, in addition to facial gear, such as goggles for safety precaution.
  5. Pipet the appropriate amount of ultra pure water into your primer containers. Coat the entire bottom of the primer container with ultra pure water. Make sure to mix the primer and the ultra pure water evenly by pipetting up and down.
  6. Now that your primers are diluted appropriately, take 10 microliters from the primer container and transfer it into a 1.5 microcentrifuge tube that is appropriately labelled with your initials, the date, the primer letter, as well as its concentration, which will be 25 micromoles.
  7. Pipet 90 microliters of ultra pure water into the 1.5 microcentrifuge tube that contains your 10 microliters of 250 micromoles primer. Make sure to mix the diluted primer and the ultra pure water evenly by pipetting up and down.
  8. Now your primers are in working condition, and it has a concentration of 25 micromoles. Store your original concentration primers and 250 micromoles diluted primers in the -20 degrees Celsius refrigerator.
  9. Remove your personal protective equipment, store away the ultra pure water, and put the micropipet into its appropriate condition.
  10. Sanitize your lab bench and wash your hands properly.
 

Emily Cao
June 2015
Cui Lab
Washington University in St. Louis
 

Protocol: Making New Gel
 

Objective: The new gel is an incredibly important aspect of the mutagenesis experiment protocol. It is commonly utilized throughout the first polymerase chain reaction, second polymerase chain reaction, and must be run in fresh 1X TAE buffer. Knowing how to make new gel is also a useful skill to be functional within a laboratory.
 

Materials:
  • 0.8 grams agarose powder
  • 100 milliliters 1X TAE Buffer
  • 5 microliters Ethidium Bromide (Carcinogen, extremely toxic. Be careful)
  • 10 microliter Pipet
  • Saran Wrap
  • Aluminum Foil
  • Laboratory Coat
  • Gloves
  • Protective Facial Gear, i.e. Goggles (Optional)
  • Microwave
  • Hot Hand Protector
  • Graduated Cylinder
  • KimTech Wipes
  • Silver Spoon
  • Brush
  • Weight
  • Wax Paper OR Plastic Boat
  • Gel Rack
  • Gel Tray
  • Large Gel Well Teeth
  • New Gel Glass Container
 

Procedure:
 
  1. Put on your personal protective equipment, such as your laboratory coat, gloves, and protective facial gear, such as laboratory goggles.
  2. Zero the weight by placing either a piece of wax paper or a plastic boat and pressing the bottom right button. Start by measuring 0.8 grams of agarose powder by transferring from its original container and onto the weight with the utilization of the silver spoon.
  3. The agarose powder is on the shelf above the lab bench in alphabetical order toward the left, whereas the wax paper or the plastic boat is in the drawer across from the weight. Make sure to brush off any leftover agarose powder from the table into a Fisherbrand autoclave bag.
  4. Pour the 0.8 grams of agarose powder into a glass container specifically used for making new gel. This can be found on the shelf located above the microwave.
  5. Measure 100 milliliters of 1X TAE Buffer from the plastic container on the side of the sink into the graduated cylinder. Ensure the volume by placing the graduated cylinder on a flat surface at eye level.
  6. Pour the 100 milliliters of 1X TAE Buffer into the new gel container with the 0.8 grams agarose powder. Mix evenly by gently shaking the container so that the agarose powder is incorporated into the 1X TAE Buffer.
  7. Using the 10 microliter Pipet, transfer 5 microliters of ethidium bromide into the new gel container and shake carefully in order to mix it evenly into the 100 milliliters 1X TAE Buffer and the 0.8 grams agarose powder.
  8. Place several KimTech Wipes in the opening of the bottle and microwave in 30 second intervals and check in between to see that the solution is not boiling.
  9. During the microwave session, place two large gel trays into a gel rack. Make sure that it is flatly pinned to the bottom. Secure two sets of large gel well teeth toward the top of the gel tray. Place this structure into the 4 degree Celsius refrigerator.
  10. Remove the new gel container from the container with the hot hand protector and pour an even amount of the solution into each of the two large gel trays in the gel rack inside of the 4 degree Celsius refrigerator.
  11. Close the 4 degree Celsius refrigerator and wait for approximately 30 minutes. You may check in 10 minute intervals to see if the gel has solidified appropriately.
  12. Once the gel has solidified properly, remove it from the 4 degree Celsius refrigerator. Take out the large gel well teeth sets.
  13. Wrap the newly made gel in a layer of saran wrap including the gel boat. Wrap once more in a layer of aluminum foil. The saran wrap maintains the new gel’s moisture, as it is sensitive to dryness, and the aluminum foil maintains the new gel’s darkness, as it is sensitive to light.
  14. Place an appropriately labelled piece of tape with the type of gel in the container, the size of its wells, your name, the date it was created, and its purpose, and store in the 4 degrees Celsius refrigerator.
  15. Remove your personal protective equipment and sanitize your lab bench.
  16. Wash your hands thoroughly.
Objective: The diagnostic gel is an extraordinarily significant aspect of the mutagenesis experiment protocol. It is commonly utilized throughout the MiniPrep procedure, and does not have to be run in fresh 1X TAE buffer. Knowing how to make diagnostic gel is also a rudimentary skill to be useful within a laboratory.
 

Materials:
  • 0.8 grams agarose powder
  • 100 milliliters 1X TAE Buffer
  • 5 microliters Ethidium Bromide (Carcinogen, extremely toxic. Be careful)
  • 10 microliter Pipet
  • Saran Wrap
  • Aluminum Foil
  • Laboratory Coat
  • Gloves
  • Protective Facial Gear, i.e. Goggles (Optional)
  • Microwave
  • Hot Hand Protector
  • Graduated Cylinder
  • KimTech Wipes
  • Silver Spoon
  • Brush
  • Weight
  • Wax Paper OR Plastic Boat
  • Gel Rack
  • Gel Tray
  • Small Gel Well Teeth
  • Diagnostic Gel Erlenmeyer Container
 

Protocol: Making Diagnostic Gel
 
  1. Put on your personal protective equipment, such as your laboratory coat, gloves, and protective facial gear, such as laboratory goggles.
  2. Zero the weight by placing either a piece of wax paper or a plastic boat and pressing the bottom right button. Start by measuring 0.8 grams of agarose powder by transferring from its original container and onto the weight with the utilization of the silver spoon.
  3. The agarose powder is on the shelf above the lab bench in alphabetical order toward the left, whereas the wax paper or the plastic boat is in the drawer across from the weight. Make sure to brush off any leftover agarose powder from the table into a Fisherbrand autoclave bag.
  4. Pour the 0.8 grams of agarose powder into a glass container specifically used for making new gel. This can be found on the shelf located above the microwave.
  5. Measure 100 milliliters of 1X TAE Buffer from the plastic container on the side of the sink into the graduated cylinder. Ensure the volume by placing the graduated cylinder on a flat surface at eye level.
  6. Pour the 100 milliliters of 1X TAE Buffer into the diagnostic gel Erlenmeyer flask with the 0.8 grams agarose powder. Mix evenly by gently shaking the container so that the agarose powder is incorporated into the 1X TAE Buffer.
  7. Using the 10 microliter Pipet, transfer 5 microliters of ethidium bromide into the Erlenmeyer flask and shake carefully in order to mix it evenly into the 100 milliliters 1X TAE Buffer and the 0.8 grams agarose powder.
  8. Place several KimTech Wipes in the opening of the bottle and microwave in 30 second intervals and check in between to see that the solution is not boiling.
  9. During the microwave session, place two large gel trays into a gel rack. Make sure that it is flatly pinned to the bottom. Secure two sets of small gel well teeth toward the top of the gel tray. Place this structure into the 4 degree Celsius refrigerator.
  10. Remove the new gel container from the container with the hot hand protector and pour an even amount of the solution into each of the two large gel trays in the gel rack inside of the 4 degree Celsius refrigerator.
  11. Close the 4 degree Celsius refrigerator and wait for approximately 30 minutes. You may check in 10 minute intervals to see if the gel has solidified appropriately.
  12. Once the gel has solidified properly, remove it from the 4 degree Celsius refrigerator. Take out the small gel well teeth sets.
  13. Wrap the newly made gel in a layer of saran wrap including the gel boat. Wrap once more in a layer of aluminum foil. The saran wrap maintains the new gel’s moisture, as it is sensitive to dryness, and the aluminum foil maintains the new gel’s darkness, as it is sensitive to light.
  14. Place an appropriately labelled piece of tape with the type of gel in the container, the size of its wells, your name, the date it was created, and its purpose, and store in the 4 degrees Celsius refrigerator.
  15. Remove your personal protective equipment and sanitize your lab bench.
  16. Wash your hands thoroughly.
 

Protocol: Polymerase Chain Reaction 1
 

Objective: Now that you have designed your DNA mutations and diluted your primers into working condition, it is time to start your first polymerase chain reaction. This first polymerase chain reaction will generate your PCR products of AB and CD.
 

Materials:
  • 68 microliters dH2O
  • 10 microliters 10X Pfu Buffer
  • 5 microliters DMSO
  • 5 microliters dNTP (4 millimoles working stock)
  • 2 microliters Primer A (25 micromoles side primer 1)
  • 2 microliters Primer B (25 micromoles mutation primer 1)
  • 2 microliters Primer C (25 micromoles mutation primer 2)
  • 2 microliters Primer D (25 micromoles side primer 2)
  • 2 microliters template DNA (0.1 micromoles)
  • 1 microliter cloned Pfu (Strategene)
  • 10 microliters dye
  • Ladder
  • PCR Tubes for every PCR product
  • PCR Machine
  • New Gel
  • Gel Blade
  • Weight
  • Ultraviolet Light
  • Calculator
  • 1.5 Microcentrifuge Tube for every PCR 1 product
  • Gel Electrophoresis Box
  • Computer
  • Carestream MI Application
  • Carestream Gel Logic Model 112 Machine
  • Saran Wrap
  • 200 microliter Pipet
  • 10 microliter Pipet
  • Laboratory Coat
  • Gloves
  • Facial Protection Gear, i.e. Goggles (Mandatory)
 

Procedure:
 
  1. Put on your personal protective equipment (Laboratory coat, gloves, and facial protection gear) and proceed toward your lab bench.
  2. For your PCR product AB, use a 200 microliter pipet to transfer 34 microliters of dH2O into a PCR tube. These are located in the white cardboard box on the shelf above the lab bench.
  3. With the utilization of a 10 microliter pipet, combine 5 microliters 10x Pfu buffer, 2.5 microliters DMSO, 2.5 microliters DNTP (4 millimoles working stock), 2 microliters of Primer A (25 micromoles side primer 1), 2 microliters of Primer B (25 micromoles mutation primer 1), and 1 microliter of template DNA (0.1 micromoles) in the PCR tube.
  4. For each new component you incorporate, you must remember to mix thoroughly. Pipet up and down in each tube that you source from, and pipet up and down once you place it into the PCR tube so that it is evenly added into the solution.
  5. For your PCR product CD, use a 200 microliter pipet to transfer 34 microliters of dH2O into a PCR tube. These are located in the white cardboard box on the shelf above the lab bench.
  6. With the utilization of a 10 microliter pipet, combine 5 microliters 10x Pfu buffer, 2.5 microliters DMSO, 2.5 microliters DNTP (4 millimoles working stock), 2 microliters of Primer D (25 micromoles side primer 1), 2 microliters of Primer C (25 micromoles mutation primer 1), and 1 microliter of template DNA (0.1 micromoles) in the PCR tube.
  7. Remove the container of the cloned Pfu (Strategene) from the -20 degrees Celsius refrigerator and store it in a cold box. The reason why enzymes must be kept in this container is due to the fact that they degrade extremely easily and also because they are expensive, and must be maintained in this stable condition.
  8. Pipet 1 microliter of the cloned Pfu (Strategene) into your PCR product AB. Mix evenly.
  9. Pipet 1 microliter of the cloned Pfu (Strategene) into your PCR product CD. Mix evenly.
  10. Place your PCR products into the PCR machine. Select the “Pfu” option due to the fact that this is the enzyme that will be carrying out the polymerase chain reaction-- it will be creating the complementary nucleotides on the template DNA strand you provided.
  11. Check to see that the settings meet these requirements:
    1. 1. 95 degrees Celsius to 98 degrees Celsius for 2 minutes.
    2. 2. 95 degrees Celsius to 98 degrees Celsius for 45 seconds.
    3. 3. 45 degrees Celsius to 48 degrees Celsius for 45 seconds.
    4. 4. 72 degrees Celsius for 2 minutes.
    5. 5. 72 degrees Celsius for 10 minutes.
    6. 6. A maintained stage at 4 degrees Celsius.
      1. The PCR machine’s job is to create a setting in which the reaction can be carried out in its ideal temperature.
  12. After the amount of time specified on the PCR machine for the reaction to take, remove your PCR products from the machine and set them in a rack on your lab bench. There should be 50 microliters of solution in each PCR product tube.
  13. Pipet 5 microliters of dye into PCR product AB using the 10 microliter pipet.
  14. Pipet 5 microliters of dye into PCR product CD using the 10 microliter pipet.
  15. Place a new gel with large wells into the gel electrophoresis box. The new gel should have the amount of wells in which the total amount of PCR products as well as the ladder have space. Ensure that the 1X TAE Buffer inside it is fresh. If not, pour it into the appropriate unwanted waste container so that it can be removed by Environmental Health & Safety. Load the gel electrophoresis box with new 1X TAE Buffer. Pop any air bubbles.
  16. Using a 200 microliter pipet, take 55 microliters of your PCR product dye mixture. Pipet up and down so that it is mixed evenly, and then load it into the proper wells. Each PCR product should now be appropriately loaded.  
  17. Pipet 10 microliters of your ladder into the small well located toward the side of the gel. Mix evenly before doing so.
  18. Put on the lid of the gel electrophoresis box, and run for approximately 15-20 minutes. Check in 5 minute intervals to check whether or not the DNA has run toward the third line of the boat tray that holds the gel.
  19. During gel electrophoresis, open the Carestream Gel Logic Model 112 machine, and place saran wrap on its surface. Make sure that the saran wrap is evenly spread out and that there are no air bubbles and creases-- these disturbances may interfere with the photograph of your gel.
  20. After gel electrophoresis, place your gel into the Carestream Gel Logic Model 112 machine and lose the door. Turn on the ultraviolet light, and open the Carestream MI Application on your computer. Select the “Capture GL 112” option and then choose “Preview” and then select “Capture”. Invert the photograph and make sure the picture is unsaturated so that the lanes look a lot more clear.
  21. Store the picture into the image database, and the experiment “PCR 1” with the type as “New Gel”. Edit the description so that the amount of time the gel ran is recorded as well as the lanes associated with the PCR products in addition to the location of the ladder.
  22. Print the photograph and tape it into your laboratory notebook. Label the lanes with the names of the PCR products as well as the ladder.
  23. Take out 1.5 microcentrifuge tubes for each PCR product you have. Label them accordingly with your initials, the date and the name of the PCR product.
  24. Weigh each 1.5 microcentrifuge tube and record its mass in grams in your notebook. Write down the mass on the side of each tube.
  25. Bring the gel into the ultraviolet light machine in the back room. Close the curtain, make sure you have on your protective facial gear, and turn on the ultraviolet light. The ethidium bromide within the gel reacts strongly to ultraviolet light, and serves as an indicator for the location of your mutation DNA.
  26. Using the gel blade, carefully excise the mutation DNA from the gel and place it into the respective 1.5 microcentrifuge tube.
  27. Turn off the ultraviolet light and turn on the back room light.
  28. Weigh your 1.5 microcentrifuge tubes containing your excised DNA gel. Record the mass in grams in your laboratory notebook as well as the side of the tube.
  29. Subtract the mass of the 1.5 microcentrifuge tube from the mass of the 1.5 microcentrifuge tube containing the excised DNA gel. The difference is the mass of the excised DNA gel. Record that mass in grams in your laboratory notebook as well as the side of the tube.
  30. Store the 1.5 microcentrifuge tubes containing the excised DNA gels in the 4 degrees Celsius refrigerator.
  31. Sanitize your lab bench, and remove your personal protective equipment.
  32. Wash your hands thoroughly.
 

Protocol: Polymerase Chain Reaction 2
 

Objective: The second polymerase chain reaction is another exceptionally important piece to the progress of the procedure. Its primary goal is to create the PCR product acknowledged as AD by combining the AB and CD PCR products. This is then utilized throughout further aspects of the mutagenesis protocol.
 

Materials:
  • 62 microliters dH2O
  • 10 microliters 10X Pfu Buffer
  • 5 microliters DMSO
  • 5 microliters dNTP (4 millimoles working stock)
  • 4 microliters Primer D (25 micromoles side primer 2)
  • 4 microliters Primer A (25 micromoles side primer 1)
  • 4 microliters PCR-AB
  • 4 microliters PCR-CD
  • 2 microliters cloned Pfu (Strategene)
  • 200 microliter Pipets
  • 10 microliter Pipets
  • Laboratory Coat
  • Gloves
  • Protective Facial Gear, i.e. Lab Goggles (Optional)
  • PCR Tubes for every PCR 2 Product
  • PCR Machine
 

Procedure:
 
  1. Put on your personal protective equipment, such as your laboratory coat, gloves, and your protective facial gear, i.e. lab goggles.
  2. Prepare a PCR tube for every PCR 2 product, and make sure to remove your PCR tubes containing your PCR 1 products from the -20 degrees refrigerator. Ensure that they are labelled properly with your name, the date, the title of your PCR products, and the step you are currently at within your experiment.
  3. Using the 200 microliter Pipet, transfer 62 microliters of ultra pure water into each respective PCR tube that will contain your PCR 2 product.
  4. Using the 10 microliter Pipet, incorporate 10 microliters 10X Pfu Buffer, 5 microliters DMSO, 5 microliters dNTP, 4 microliters Primer D (25 micromoles side primer 2), 4 microliters Primer A (25 micromoles side primer 1), 4 microliters PCR-AB, and 4 microliters PCR CD into the PCR tubes that will contain your PCR 2 product.
  5. Remove the container that stores the cloned Pfu (Strategene) from the -20 degrees Celsius refrigerator and place in a cold box. The reason why enzymes must be stored at such a low temperature is due to the fact that they denature extremely easily and are also really expensive, so they must be kept in a stable state within a cold box.
  6. Using the 10 microliter, transfer 2 microliters of the cloned Pfu (Strategene) into the PCR  tubes that will contain your PCR 2 products.
  7. Place the PCR tubes into the PCR machine and select the “Pfu” option. Make sure that the PCR machine follows this specified setting:
    1. 1. 95-98 degrees Celsius for 2 minutes
    2. 2. 95-98 degrees Celsius for 20 seconds
    3. 45-48 degrees Celsius for 20 seconds
    4. 72 degrees Celsius for 15 seconds/kb
    5. 72 degrees Celsius for 3 minutes
    6. A maintained temperature at 4 degrees Celsius
      1. The PCR machine’s job is to create a setting in which the reaction can be carried out in its ideal temperature.
 

Protocol: Cut Vector & Polymerase Chain Reaction 2 With Same Two Enzymes
 

Objective: The purpose behind the cut vector protocol is so that we can put purified PCR 2 products into the wild type’s plasmid DNA. In order to do this, we must cut the PCR 2 product in addition to the wild type’s plasmid DNA with the restriction enzymes. Once the restriction enzymes have executed their cuts on the PCR 2 product, we now have an “insert”. Afterwards, the plasmid DNA of the wild-type will also be cut with the restriction enzymes. Subsequently, we run a gel with the cut wild-type plasmid DNA. We will usually have one big band and one small band. We will then cut out the big band and purify it. The purified band of the wild-type plasmid DNA is then referred to as the vector. Overall, the objective of this protocol is to prepare the “insert” and the “vector” for the ligation procedure.
 

Materials:
 
  • Purified PCR 2 Products (Mutation DNA Sequence)
  • Wild-Type Plasmid (Wild Type DNA Sequence)
  • 1.5 Microcentrifuge Tubes
  • 0.6 Microcentrifuge Tubes
  • Ultra Pure Water
  • 10 microliter Pipet
  • 20 microliter Pipet
  • Choice Between Buffer 1.1, Buffer 2.1, Buffer 3.1, OR CutSmart Buffer
  • Restriction Enzyme #1
  • Restriction Enzyme #2
  • Laboratory Coat
  • Globes
  • Protective Facial Gear, i.e. Lab Goggles (Optional)
 

Procedure:
 
  1. Put on your personal protective equipment, such as your laboratory coat, gloves, and your protective facial gear, i.e. lab goggles.
  2. Before you do anything, make sure to check the compatibility of 2 or more enzymes when used together, such as their incubation temperature as well as their desired buffer.
  3. Check for specific potential problems, especially for complications such as methylation.
  4. Decide the unit size of the enzymes necessitated (1 ug DNA needs 1 unit of enzyme)
  5. Find out the total volume of enzymes needed according to the enzyme concentration (For example: 10 units/microliter).
  6. Discover the type of buffer the restriction enzymes prefer and whether or not they need BSA.
  7. Decide the volume of the reaction. It should be at least 10X the total volume of the enzymes needed. (DNA volume + Buffer volume [0.1X total volume) + BSA volume (0.1 or 0.01X total volume) + Enzyme volume).
  8. Calculate the amount of ultra pure water needed to be added (Total volume-DNA-Buffer-BSA-Enzyme).
  9. Add the ingredients in the following order:
    1. dH2O
    2. DNA
    3. Buffer
    4. BSA
    5. Restriction Enzymes
  10. Prepare 2 1.5 microcentrifuge tubes for each purified PCR 2 product you have within your possession.
  11. Using a 20 microliter pipet, transfer your calculated volume of ultra pure water into each 1.5 microcentrifuge tube.
  12. Using a 10 microliter Pipet, transfer your calculated volume of your purified PCR 2 product into its respective 1.5 microcentrifuge tube.
  13. Using a 10 microliter Pipet, transfer your calculated volume of your wild-type plasmid DNA into its respective 1.5 microcentrifuge tube.
  14. Using a 10 microliter Pipet, transfer the 2 microliters of your appropriate buffer (Whether it is Buffer 1.1, Buffer 2.1, Buffer 3.1, or CutSmart Buffer) into both the 1.5 microcentrifuge tubes.
  15. Remove the containers of the Restriction Enzyme #1 as well as the Restriction Enzyme #2 from the -20 degrees Celsius refrigerator and store it in a cold box. The reason why enzymes must be kept in this container is due to the fact that they degrade extremely easily and also because they are expensive, and must be maintained in this stable condition.
  16. Using a 10 microliter Pipet, transfer your calculated volume microliter of both your Restriction Enzyme #1 and your Restriction Enzyme #2 into both the 1.5 microcentrifuge tubes.
  17. Incubate the reaction at its specified digestion temperature (Usually 37 degrees Celsius) for approximately 1 hour.
  18. Stop the digestion by heat inactivation (65 degrees Celsius for 15 minutes).
  19. Pipet the correlating volume of dye into the 1.5 microcentrifuge tube containing your digested wild-type plasmid DNA.
  20. You may now store your 1.5 microcentrifuge tubes containing your cut and purified PCR 2 products in the -20 degrees Celsius refrigerator.
  21. Place a new gel with small wells into the gel electrophoresis box. The new gel should have the amount of wells in which the total amount of digested wild-type DNA  products as well as the ladder have space. Ensure that the 1X TAE Buffer inside it is fresh. If not, pour it into the appropriate unwanted waste container so that it can be removed by Environmental Health & Safety. Load the gel electrophoresis box with new 1X TAE Buffer. Pop any air bubbles.
  22. Using a 200 microliter pipet, take the correlating volume of your digested wild-type plasmid DNA and dye mixture. Pipet up and down so that it is mixed evenly, and then load it into the proper wells. Each digested wild-type DNA  product should now be appropriately loaded.  
  23. Pipet 10 microliters of your ladder into the small well located toward the side of the gel. Mix evenly before doing so.
  24. Put on the lid of the gel electrophoresis box, and run for approximately 15-20 minutes. Check in 5 minute intervals to check whether or not the DNA has run toward the third line of the boat tray that holds the gel.
  25. During gel electrophoresis, open the Carestream Gel Logic Model 112 machine, and place saran wrap on its surface. Make sure that the saran wrap is evenly spread out and that there are no air bubbles and creases-- these disturbances may interfere with the photograph of your gel.
  26. After gel electrophoresis, place your gel into the Carestream Gel Logic Model 112 machine and lose the door. Turn on the ultraviolet light, and open the Carestream MI Application on your computer. Select the “Capture GL 112” option and then choose “Preview” and then select “Capture”. Invert the photograph and make sure the picture is unsaturated so that the lanes look a lot more clear.
  27. Store the picture into the image database, and the experiment “Cut Vector & PCR 2 With The Same Two Enzymes” with the type as “New Gel”. Edit the description so that the amount of time the gel ran is recorded as well as the lanes associated with the digested wild-type DNA  products in addition to the location of the ladder.
  28. Print the photograph and tape it into your laboratory notebook. Label the lanes with the names of the digested wild-type DNA  products as well as the ladder.
  29. Take out 1.5 microcentrifuge tubes for each digested wild-type DNA  product you have. Label them accordingly with your initials, the date and the name of the digested wild-type DNA  product.
  30. Weigh each 1.5 microcentrifuge tube and record its mass in grams in your notebook. Write down the mass on the side of each tube.
  31. Bring the gel into the ultraviolet light machine in the back room. Close the curtain, make sure you have on your protective facial gear, and turn on the ultraviolet light. The ethidium bromide within the gel reacts strongly to ultraviolet light, and serves as an indicator for the location of your mutation DNA.
  32. Using the gel blade, carefully excise the mutation DNA from the gel and place it into the respective 1.5 microcentrifuge tube.
  33. Turn off the ultraviolet light and turn on the back room light.
  34. Weigh your 1.5 microcentrifuge tubes containing your excised DNA gel. Record the mass in grams in your laboratory notebook as well as the side of the tube.
  35. Subtract the mass of the 1.5 microcentrifuge tube from the mass of the 1.5 microcentrifuge tube containing the excised DNA gel. The difference is the mass of the excised DNA gel. Record that mass in grams in your laboratory notebook as well as the side of the tube.
  36. Identify the largest mass, and multiply that amount by 1000. Round this amount to its nearest hundredth place. This number is the volume of membrane binding solution you will be pipetting into each tube.
  37. Pipet the volume calculated of membrane binding solution into each tube with the utilization of the 1000 microliter Pipet. Tap the 1.5 microcentrifuge tubes on the lab bench in order to make sure all of the contents are evenly incorporated.
  38. Turn on the heat block and place the 1.5 microcentrifuge tubes containing the excised DNA from the gel in addition to the membrane binding solution into each slot.
  39. While the 1.5 microcentrifuge tubes are on the heat block, prepare a spin column in a collection tube for each digested wild-type DNA  product involved throughout your experiment. Label the spin column and collection tube structure appropriately, with your initials, the date, the name of your digested wild-type DNA  product, in addition to the step you are at within your protocol.
  40. After approximately 10 minutes, remove the 1.5 microcentrifuge tubes from the heat block and transfer the solutions inside them into each spin column and collection tube structure. Centrifuge for about 1 minute at full speed.
  41. Discard the flow-through inside the collection tubes and place the spin columns inside the original 1.5 microcentrifuge tubes. Now, it is the point within the experiment in which the spin columns must be taken to the vacuum to wash out any excess salts and buffers from the membrane binding solution.
  42. Attach a clean adapter onto each port on the vacuum that correlates with your digested wild-type DNA  product. Put on each spin column that should contain your digested wild-type DNA  product onto the clean adapter. Turn the blue handle so that it sits in a vertical position, and then turn on the vacuum by twisting the black knob above it in a counter-clockwise direction.
  43. Take 750 microliters of the membrane washing solution for PCR purification and pipet into each spin column.
  44. Take 500 microliters of the membrane washing solution for PCR purification and pipet into each spin column after all of the membrane washing solution from the previous round has been drained.
  45. Turn off the vacuum by twisting the black knob located above it in a clockwise direction and turning the blue handle so that it sits in a horizontal position. Remove the clean adapters and then place them onto the brown paper towel next to the vacuum so that they can dry. Place the spin columns back into their respective 1.5 microcentrifuge tubes.
  46. Centrifuge the spin columns in their 1.5 microcentrifuge tubes for one minute at full speed.
  47. Discard the flow-through from the 1.5 microcentrifuge tubes and then place the spin columns back into the original 1.5 microcentrifuge tubes. Centrifuge for another minute at full speed.
  48. During the centrifuge process, label new 1.5 microcentrifuge tubes appropriately with your name, the date, the titles of the digested wild-type DNA  products, in addition to the step you are currently at within your experiment. Discard the original 1.5 microcentrifuge tubes after the centrifuge process and load them with their respective spin columns.
  49. In order to elute the DNA, use your 200 microliter pipet to transfer 50 microliters of nuclease-free water into each of your digested wild-type DNA  products. Centrifuge this for one minute at full speed.
  50. Make sure that you are utilizing the centrifuge appropriately by ensuring that it is balanced with tubes containing equivalent volumes located across from each other at all times and that the plastic cover is secured on tightly throughout the process.
  51. The solution at the bottom of your new 1.5 microcentrifuge tubes is your purified digested wild-type DNA  from the cut vector restriction enzyme digestion. You may now discard the spin columns into the proper Fisherband autoclave bags.
  52. Prepare 0.6 microcentrifuge tubes for each digested wild-type DNA  product and label them appropriately with your name, the date, the title of the digested wild-type DNA  products, and the step at which you are at within your experiment.
  53. Using the 10 microliter Pipet, transfer 5 microliters of ultra pure water into each 0.6 microcentrifuge tube.
  54. Using the 10 microliter Pipet, transfer 3 microliters of loading dye into each 0.6 microcentrifuge tube.
  55. Using the 10 microliter Pipet, take 2 microliters of digested wild-type DNA  from your new 1.5 microcentrifuge tubes and transfer them into the loading dye and ultra pure water mixture into your new 0.6 microcentrifuge tubes.
  56. Load a diagnostic gel with small wells into the gel electrophoresis box-- the 1X TAE Buffer does not need to be exchanged.
  57. Pipet 10 microliters of the ladder into the side well, and then follow through with 10 microliters of each of the digested wild-type DNA  product, loading dye, and ultra pure water mixture into the subsequent wells.
  58. During gel electrophoresis, place a layer of saran wrap on the surface of the Gel Logic Model 112 Carestream machine. Afterwards, transfer the diagnostic gel into the machine, close the door, and turn on the ultraviolet light.
  59. Open the Carestream MI Application in your computer and select “Capture GL 112” and then choose “Preview” and then click “Capture”. Invert the desaturate the photograph in order to make the picture more clear. Print the picture out in order to tape and label the lanes within your laboratory notebook, and store the photograph inside of your image database.
  60. Edit the description with the amount of time you ran the diagnostic gel for, which would be approximately 15 minutes, as well as the contents of each of the wells and the lanes. Make sure the type is “Diagnostic Gel” and that the experiment is “Cut Vector Purification”.
  61. Remove your personal protective equipment and sanitize your lab bench.
  62. Wash your hands thoroughly.
  63. Sanitize your lab bench, and remove your personal protective equipment.
  64. Wash your hands thoroughly.
 

Protocol: Ligation Product Creation
 

Objective: The purpose behind ligation is so that the “insert” and the “vector” can be combined together so that they can form a plasmid to be shocked into competent cells throughout electroporation. This step is an imperative piece to the mutagenesis protocol, and is also an important preparation for the transformation experiment.
 

If the concentration is high enough:
 

Materials:
 
  • 0.5 microliters vector
  • 4 microliters insert (Cut and purified PCR 2 products)
  • 5 microliters fast ligation buffer (Promega)
  • 0.5 microliters T4 ligase (Promega)
  • 10 microliter Pipet
  • 2.5 microliter Pipet
  • 0.6 microcentrifuge tubes
  • Laboratory Coat
  • Gloves
  • Protective facial gear, i.e. lab goggles
  • 18 degrees Celsius Incubator
 

Procedure:
 
  1. Prepare a 0.6 microcentrifuge tube for each ligation product. Label accordingly with your name/initials, the date, the name of your purified and cut PCR 2 product (The insert), the name of your cut and purified wild-type plasmid DNA (The vector), and the step you are currently at within your experiment (The ligation).
  2. Using a 2.5 microliter Pipet, transfer 0.5 microliters of your vector into the 0.6 microcentrifuge tube.
  3. Using a 10 microliter Pipet, transfer 4 microliters of your 10X ligation buffer into the 0.6 microcentrifuge tube.
  4. Using a 10 microliter Pipet, transfer 5 microliters of your insert into the 0.6 microcentrifuge tube.
  5. Remove the containers of the T4 ligase from the -20 degrees Celsius refrigerator and store it in a cold box. The reason why enzymes must be kept in this container is due to the fact that they degrade extremely easily and also because they are expensive, and must be maintained in this stable condition.
  6. Using a 2.5 microliter Pipet, transfer 0.5 microliters of the T4 ligase into the 0.6 microcentrifuge tube.
  7. There should be a volume of 10 microliters within your total reaction.
  8. Store this mixture in an 18 degrees Celsius incubator or at room temperature for 5 minutes or more. The longer the time will ensure higher efficiency. Normally, you should incubate your ligation product at 1-2 hours.
 

OR
 

If the concentration is too low:
 

Materials:
  • 0.5 microliters vector
  • 8 microliters insert (Cut and purified PCR 2 products)
  • 1 microliter T4 10X Ligation Buffer (Promega)
  • 0.5 microliters T4 ligase (Promega)
  • 10 microliter Pipet
  • 2.5 microliter Pipet
  • 0.6 microcentrifuge tubes
  • Laboratory Coat
  • Gloves
  • Protective facial gear, i.e. lab goggles
  • 18 degrees Celsius Incubator  
 
  1. Prepare a 0.6 microcentrifuge tube for each ligation product. Label accordingly with your name/initials, the date, the name of your purified and cut PCR 2 product (The insert), the name of your cut and purified wild-type plasmid DNA (The vector), and the step you are currently at within your experiment (The ligation).
  2. Using a 2.5 microliter Pipet, transfer 0.5 microliters of your vector into the 0.6 microcentrifuge tube.
  3. Using a 10 microliter Pipet, transfer 8 microliters of your insert into the 0.6 microcentrifuge tube.
  4. Using a 10 microliter Pipet, transfer 1 microliters of your fast ligation buffer into the 0.6 microcentrifuge tube.
  5. Remove the containers of the T4 ligase from the -20 degrees Celsius refrigerator and store it in a cold box. The reason why enzymes must be kept in this container is due to the fact that they degrade extremely easily and also because they are expensive, and must be maintained in this stable condition.
  6. Using a 2.5 microliter Pipet, transfer 0.5 microliters of the T4 ligase into the 0.6 microcentrifuge tube.
  7. There should be a volume of 10 microliters within your total reaction.
  8. Store this mixture in an 18 degrees Celsius incubator overnight.
 

Protocol: Preparation for Transformation
 

Objective: Before you begin the transformation protocol, you must take some steps in order to prepare for it. These are extremely important, and if they are not done correctly, your ligation product will not be properly transformed. It is also a significant component to the mutagenesis experiment.
 

Materials:
 
  • Agarose Plate for Each Ligation Product
  • Cuvette for Each Ligation Product
  • 1.5 Microcentrifuge Tube
  • Laboratory Coat
  • Gloves
  • Protective Facial Gear, i.e. Lab Goggles
 
  1. Put on your personal protective equipment, such as your laboratory coat, gloves, protective facial gear, i.e. lab goggles.
  2. Prepare a 1.5 microcentrifuge tube for each ligation product under your possession, and label accordingly with your name/initials, the date, the title of your ligation product, the name of your vector, the name of your insert, and the step you are currently at within your experiment.
  3. Remove the agarose plates from the 4 degree refrigerator and set them out at 18 degrees Celsius or room temperature in order to thaw out.
  4. Wash out your cuvettes using the solutions throughout this order:
    1. 30% Bleach
    2. Tap H2O
    3. dH2O
    4. Ultra Pure Water
    5. 70% Ethanol
    6. 95% Ethanol
  5. Invert on a brown paper towel in order to dry. Once dried, place in a bucket of ice and make sure no ice enters the interior of the cuvette. Cap the bucket and place next to your electroporator.
  6. Place the 0.6 microcentrifuge tubes containing your ligation products next to your electroporator.
  7. Once your agarose plates have defrosted and are out next to your electroporator in addition to your 0.6 microcentrifuge tubes containing your ligation products as well as your washed and dried cuvettes, you are now ready to start the transformation protocol.
 

Protocol: Transformation (Electroporation)
 

Objective: The purpose behind transformation is to shock the ligation product (Or the plasmid of the combined vector and insert) into competent cells. These cells are then plated on agarose so that more can be amplified through further steps within the procedure, such as MiniPrep and LargePrep. Ultimately, transformation is an important step within the experiment and is an imperative milestone in which the ligation product we have created can finally be grown on a medium platform.
 

Materials:
 
  • 1000 microliter Pipet
  • 200 microliter Pipet
  • 2.5 microliter Pipet
  • LB Stock Without Antibiotic
  • 40 Microliters Competent Cells for Each Ligation Product
  • Cuvette for Each Ligation Product
  • Agarose Plate for Each Ligation Product
  • 1.5 Microcentrifuge Tube for Each Ligation Product
  • Agarose Plating Spin Table
  • Bunsen Burner
  • Lighter
  • Electroporator
  • Laboratory Coat
  • Gloves
  • Spreader
  • Bunsen burner
  • Protective Facial Gear, i.e. Lab Goggles
 

Procedure:
 
  1. Put on your personal protective equipment, such as your laboratory coat, your gloves, and your protective facial gear, i.e. lab goggles.
  2. Take the 1.5 microcentrifuge tubes you have prepared for each of your ligation products that are already properly labelled with your name/initials, date, title of your ligation product, the name of your insert, the name of your vector, as well as the step you are currently at within your experiment.
  3. Using your 1000 microliter Pipet, transfer 500 microliters of LB stock without antibiotic into your 1.5 microcentrifuge tube.
  4. Using your 2.5 microliter Pipet, transfer 2.5 microliters of your ligation product into your 40 microliters competent cell mixture. You can melt the competent cell mixture by swirling your tip, pipetting up and down, and warming it with your body temperature by holding it.
  5. Using your 200 microliter Pipet, transfer 42.5 microliters of your ligation product and competent cells mixture into your cleaned cuvette. Pipet up and down to mix, and then insert the correct direction into the electroporator.
  6. Pulse at 2500 volts twice. Listen for a “popping” sound that will indicate the electricity has destroyed your cell culture. If there is no noise, remove the cuvette from the electroporator.
  7. Once you have removed your cuvette from the electroporator, use your 200 microliter Pipet to transfer approximately 50 microliters of the LB stock without antibiotic into your cuvette. Pipet up and down, and then transfer the LB stock without antibiotic and competent cell containing the plasmid ligation product mixture into the 1.5 microcentrifuge tube containing the complete LB stock without antibiotic.
  8. You can place the 1.5 microcentrifuge tube containing your LB stock without antibiotic and your competent cells with the ligation product plasmid DNA into the shaker at 37 degrees Celsius in order to recover from the electroporation.
  9. While your 1.5 microcentrifuge tubes containing your LB stock without antibiotic and your competent cells with the ligation product plasmid DNA are shaking at 37 degrees Celsius, start making your spreaders.
  10. Start by turning on the Bunsen Burner. Do so by attaching the rubber tube from the Bunsen Burner to do the silver projection from the gas knob. Continue by turning on the black gas knob counter-clockwise. Turn on the lighter above the Bunsen Burner and make a flame. Take the glass tips from their metal containers and seal their top by sending them through the bottom of the flame. Make an L shape with the glass tip by moving it one inch through the top of the flame and allowing it to bend with the heat. Store your spreader in a sterile space.
  11. After your cells have finished shaking, place your agarose plate on an agarose plating spin table. Use your 200 microliter Pipet to transfer 150 microliters of your transformed product onto your agarose plate.
  12. Turn the agarose plating spin table at an expeditious plate as you use your spreader to evenly distribute your transformed product across the agarose production so that everything is uniformly spread out. Make sure to make vertical, horizontal, and circular motions with your spreader in order to further progress the distribution.
  13. You may now turn off your Bunsen Burner. Take your plate and store in the 4 degrees Celsius refrigerator until you leave the laboratory for the evening.
  14. Remove your personal protective equipment and sanitize your lab bench.
  15. Wash your hands thoroughly.
  16. Once you are about to go, place the plates in a 37 degree incubator. They should stay there for in between 12-16 hours until the next morning.
  17. When storing agarose plates, make sure to wrap them on all sides with laboratory parafilm and placing them in the 4 degrees Celsius refrigerator.
 

Protocol: MiniPrep (TELT Method)
 

Objective: MiniPrep is essentially a quick and dirty checkup in which we try to figure out if the competent cells we transformed express the mutation given from the ligation product’s plasmid DNA. We carry out a MiniPrep procedure by checking 4 colonies per mutation. It is basically a very crude observation. Hopefully, MiniPrep will indicate that at least one colony is positive for the mutation. Overall, MiniPrep is a gateway to the DNA amplification we participate in throughout LargePrep and is a fundamental eligibility requirement we must pass in order to move on to the next step within the mutagenesis experiment.
 

Materials:
  • TELT Solution
  • LB Stock with Antibiotic
  • 1:1 Phenol/Chloroform
  • 100% Ethanol, Prechilled to -20 degrees Celsius
  • TE Buffer
  • 10 mg/mL Dnase-free Rnase A (Optional)
  • 1.5 microcentrifuge tubes
  • 200 microliter Pipet
  • Microspatula
  • Laboratory Coat
  • Gloves
  • Protective facial gear, i.e. Lab Goggles (Optional)
 

Procedure:
 
  1. Put on your personal protective equipment such as your laboratory coat, your gloves, and your gloves and your protective facial gear, i.e. lab goggles.
  2. Remove the agarose plates you incubated at 37 degrees Celsius overnight and place them on your lab bench at 18 degrees Celsius, or room temperature.
  3. Prepare 4 1.5 microcentrifuge tubes for every transformed product. Label accordingly with your name/initials, the date, the name of your transformed product, the number associated with your transformed product, and the step at which you are currently at within your experiment.
  4. Using your 200 microliter Pipet, transfer 100 microliters of the TELT solution into each appropriately labelled 1.5 microcentrifuge tube.
  5. In order to isolate plasmid DNA from transformed colonies grown on agar plates, prepare the cells by using a microspatula to scoop out an entire bacterial colony grown to 2- to 5- nm in diameter from the agarose plate and into your 1.5 microcentrifuge tubes containing 100 microliters of your TELT solution.
  6. Vortex thoroughly for 5 seconds in order to suspend the cell.
  7. Add 100 microliters of your 1:1 phenol/chloroform with your 200 microliter Pipet and thoroughly vortex for 5 seconds. This mixture may be left at room temperature for less than or equal to 15 minutes. Plasmid yield will elevate with increasing duration of the incubation; however, incubation periods > 15 minutes may result in phenol-mediated modification of DNA.
  8. Microcentrifuge at 1 minute at 15,000 X g, or the maximum speed.
  9. Using your 200 microliter Pipet, carefully withdraw 75 microliters of the upper aqueous phase and transfer the contents into a clean 1.5 microcentrifuge tube. Do not agitate the resolved phases. If mixing occurs, recentrifuge. When collecting the top layer, avoid picking the debris at the interface.
  10. To the supernatant, use your 200 microliter Pipet to add 150 microliters of your chilled 100% ethanol. Mix the contents well in order to precipitate the plasmid DNA.
  11. Pellet the nucleic acids by microcentrifuging for 5 minutes at maximum speed.
  12. Discard the supernatant by inverting the tube. When all the supernatant has drained, hold the tube in the same position for a few seconds and wipe off the last droplet from the rim of the tube by touching the edge of a Kleenex paper tissue.
  13. Wash the pellet with 1 ml of cold 100% ethanol and harvest the nucleic acid pellet. After centrifugation, decant the supernatant carefully as the pellet may be loose.
  14. Cap the tube. Stab a small hole in the cap with a thumbtack or a syringe needle. Place the tube in a vacuum desiccator (without desiccant). Apply vacuum until the nucleic acid pellet appears completely dry. A water-pumped vacuum line suffices for the purpose and usually takes less than or equal to 15 minutes.
  15. Dissolve the pellet in 30 µl TE buffer. Vortex the contents well, capturing most of the DNA around the inner surface of the microcentrifuge tube. Store as in the support protocol and use 2 to 5 µl of DNA solution in a final 20-µl reaction volume for restriction digestion.
  16. Contaminating RNA may interfere with detection of DNA fragments on the agarose gel; it can be destroyed by adding 1 l of a 10 mg/ml RNase solution (DNase-free) to the digestion mixture. For scaled-up plasmid DNA preparations (He et al., 1991), increase the amounts of TELT solution and 1:1 phenol/chloroform in direct proportion to the culture volume used. For cultures ?5 ml, transfer the cells after suspension in TELT buffer into a microcentrifuge tube.
  17. Wash the final nucleic acids pellet twice with 1 ml of 100% ethanol. For cultures between 5 and 100 ml, use Corex glass tubes for treatment with TELT and phenol/chloroform and for centrifugations (Sorvall RC-5C centrifuge at 6000 X g).
 

Protocol: Restriction Digestion Subsequent to MiniPrep
 

Objective: The purpose of the restriction digestion subsequent to MiniPrep is basically to check to see if the mutation we have created is present within the gene expression of the competent cells that contain our ligation product’s plasmid DNA. We are basically going to apply the restriction enzyme we selected for it toward the beginning when we were putting together our design and comparing it with a negative control of our wild type DNA.
 

Materials:
 
  • 42 microliters Ultra Pure Water
  • Buffer 1.1, Buffer 2.1, Buffer 3.1, OR CutSmart Buffer
  • Appropriate Restriction Enzyme
  • Wild Type Plasmid DNA
  • MiniPrep Mutation Plasmid DNA
  • Laboratory Coat
  • Gloves
  • 2.5 microliter Pipet
  • 10 microliter Pipet
  • 20 microliter Pipet
  • Incubator
  • Ladder
  • Protective facial gear, i.e. Lab Goggles
  • 2 1.5 Microcentrifuge Tubes for Each Mutation
  • 2 0.6 Microcentrifuge Tubes for Each Mutation
  • 6 microliters dye
 

Procedure:
 
  1. Put on your personal protective equipment, such as your laboratory coat, your gloves, in addition to your protective facial gear, i.e. lab goggles.
  2. Prepare 2 1.5 microcentrifuge tubes for each mutation, and label appropriately with your name/initials, your date, the name of your mutation/the name of your restriction enzyme for your negative control, in addition to the step you are currently at within your experiment.
  3. You will have a final volume of 20 microliters for your restriction digestion. Use your 20 microliter Pipet to transfer approximately 11 microliters of ultra pure water into each 1.5 microcentrifuge tube.
  4. Use your 2.5 microliter Pipet to transfer 2 microliters of your appropriate buffer into your 1.5 microcentrifuge tube containing your ultra pure water.
  5. Use your 10 microliter Pipet to transfer 5 microliters of your MiniPrep mutation plasmid DNA into your 1.5 microcentrifuge tube containing your ultra pure water and your appropriated buffer.
  6. Use your 2.5 microliter Pipet to transfer 2 microliters of your appropriate buffer into your 1.5 microcentrifuge tube containing your ultra pure water.
  7. Use your 10 microliter Pipet to transfer 5 microliters of your wild type plasmid DNA into your 1.5 microcentrifuge tube containing your ultra pure water and your appropriated buffer.
  8. Remove the container of the proper restriction enzyme from the -20 degrees Celsius refrigerator and store it in a cold box. The reason why enzymes must be kept in this container is due to the fact that they degrade extremely easily and also because they are expensive, and must be maintained in this stable condition.
  9. Use your 2.5 microliter Pipet to transfer 2 microliters of your restriction enzyme into your 1.5 microcentrifuge tube containing your ultra pure water, your appropriated buffer, and your MiniPrep mutation plasmid DNA.
  10. Use your 2.5 microliter Pipet to transfer 2 microliters of your restriction enzyme into your 1.5 microcentrifuge tube containing your ultra pure water, your appropriated buffer, and your wild type plasmid DNA.
  11. Place your 1.5 microcentrifuge tubes containing your ultra pure water, your appropriated buffer, your MiniPrep mutation plasmid DNA, and your ultra pure water, your appropriate buffer, and your wild type plasmid DNA respectively into the incubator with the proper incubation temperature for your restriction enzyme for an hour.
  12. During the time period wait, make sure to prepare 2 0.6 microcentrifuge tubes for each mutation, one for the mutated plasmid DNA, and the other for the wild type plasmid DNA. Label them accordingly with your name/initials, the date, the name of your mutation or the name of your wild type plasmid DNA, and the step at which you are currently at within your experiment.
  13. Use your 10 microliter Pipet to transfer 5 microliters of ultra pure water into each 0.6 microcentrifuge tube.
  14. Use your 10 microliter Pipet to transfer 3 microliters of the loading dye into each 0.6 microcentrifuge tube.
  15. Use your 10 microliter Pipet to transfer 2 microliters of the mutated plasmid DNA into each respective 0.6 centrifuge tube as well as 2 microliters of the wild type plasmid DNA into each respective 0.6 centrifuge tube.
  16. Take a diagnostic gel with small wells and place it into a 1X TAE buffer in the gel electrophoresis box.
  17. Use your 10 microliter Pipet to transfer 10 microliters of the Ladder into the side well of the diagnostic gel.
  18. Use your 10 microliter Pipet to transfer 10 microliters of the ultra pure water, dye, and mutated plasmid DNA mixture as well as your 10 microliters of the ultra pure water, dye, and wild type plasmid DNA mixture to load into the small wells of the diagnostic gel. Remember to pipet up and down in order to blend evenly before you load into the small wells of the diagnostic gel.
  19. Put on the lid of the gel electrophoresis box, and run for approximately 15-20 minutes. Check in 5 minute intervals to check whether or not the DNA has run toward the third line of the boat tray that holds the gel.
  20. During gel electrophoresis, open the Carestream Gel Logic Model 112 machine, and place saran wrap on its surface. Make sure that the saran wrap is evenly spread out and that there are no air bubbles and creases-- these disturbances may interfere with the photograph of your gel.
  21. After gel electrophoresis, place your gel into the Carestream Gel Logic Model 112 machine and lose the door. Turn on the ultraviolet light, and open the Carestream MI Application on your computer. Select the “Capture GL 112” option and then choose “Preview” and then select “Capture”. Invert the photograph and make sure the picture is unsaturated so that the lanes look a lot more clear.
  22. Store the picture into the image database, and the experiment “Restriction Enzyme Digestion Subsequent to MiniPrep” with the type as “Diagnostic Gel”. Edit the description so that the amount of time the gel ran is recorded as well as the lanes associated with the PCR products in addition to the location of the ladder.
  23. Print the photograph and tape it into your laboratory notebook. Label the lanes with the names of the PCR products as well as the ladder.
  24. Check to see if the mutations are positive. If they all are, which they should be, begin preparing for LargePrep.
  25. Sanitize your lab bench, and remove your personal protective equipment.
  26. Wash your hands thoroughly.
 

Protocol: LargePrep (Promega SV & Miniprep Kit & Qiagen Kit)
 

Objective: The purpose behind LargePrep is basically once we know that one colony is positive for the mutation from the MiniPrep checkup, our task is to culture an enormous volume through LargePrep. This is an important piece of the mutagenesis protocol due to the fact that it provides us with a bigger bacterial yield of approximately 40-100 micrograms of DNA, and will allow us to be successful within the further pieces of our experiment.
 

Materials:
 
  • 42 microliters Ultra Pure Water
  • Buffer P1, Buffer P2, and Buffer P3
  • Buffer QBT, Buffer QC, and Buffer QF
  • LB stock with Antibiotic
  • Appropriate Restriction Enzyme
  • Wild Type Plasmid DNA
  • MiniPrep Mutation Plasmid DNA
  • Laboratory Coat
  • Gloves
  • 2.5 microliter Pipet
  • 10 microliter Pipet
  • 20 microliter Pipet
  • Incubator
  • Ladder
  • Protective facial gear, i.e. Lab Goggles
  • 2 1.5 Microcentrifuge Tubes for Each Mutation
  • 2 0.6 Microcentrifuge Tubes for Each Mutation
  • 6 microliters dye
 

Procedure:
 
  1. Night before: for each sample, make 200 ml LB and autoclave in a 1L flask. Let cool, add 200L 50 mg/ml ampicillin, add picked colony, and shake o/n @ 37oC.
  2. Pour broth into plastic centrifuge bottle. Spin for 15’ at 5000 rpm.
  3. Pour off supernatant; resuspend bacterial pellet in 10 ml Buffer P1 (this should already be cold, as its stored in the refrigerator with RNAse). Transfer resuspended pellet solution to a plastic centrifuge tube.
  4. Add 10 ml Buffer P2, mix gently, and incubate at room temperature for 5 minutes.
  5. Add 10 ml of cold Buffer P3, mix immediately but gently by inversion, and incubate on ice for 20 minutes.
  6. Spin for 30’ at 12,000 rpm @ 4 oC. During the last 10’ of the spin, equilibrate Qiagen-tip 500 column by adding 10 ml Buffer QBT. Allow column to drain.
  7. Decant supernatent 2X in Falcon 50ml tubes. Remove remaining chunks with pipet.
  8. Add decanted supernatent to column. Allow to flow through.
  9. Wash column 2X with 30 ml Buffer QC.
  10. Elute DNA by adding 2 x 5 ml Buffer QF. Catch eluate in fresh plastic centrifuge tube.
  11. Precipitate DNA with 7 ml room-temperature isopropanol. Spin for 30’ at 12,000 rpm @
  12. Pour off supernatent; add 5 ml 70% ethanol; spin 5’ at 12, 000 rpm @ 4 oC.
  13. Pour off supernatent; respin for 5’ at 12, 000 rpm @ 4 oC and remove remaining drops with vacuum.
  14. Air-dry for 5’; resuspend in 500 ml dH20 or TE, transfer to an eppendorf. Respin for 5’ at 5,000 rpm if necessary to get remaining DNA solution.
  15. Prepare 2 1.5 microcentrifuge tubes for each mutation, and label appropriately with your name/initials, your date, the name of your mutation/the name of your restriction enzyme for your negative control, in addition to the step you are currently at within your experiment.
  16. You will have a final volume of 20 microliters for your restriction digestion. Use your 20 microliter Pipet to transfer approximately 11 microliters of ultra pure water into each 1.5 microcentrifuge tube.
  17. Use your 2.5 microliter Pipet to transfer 2 microliters of your appropriate buffer into your 1.5 microcentrifuge tube containing your ultra pure water.
  18. Use your 10 microliter Pipet to transfer 5 microliters of your MiniPrep mutation plasmid DNA into your 1.5 microcentrifuge tube containing your ultra pure water and your appropriated buffer.
  19. Use your 2.5 microliter Pipet to transfer 2 microliters of your appropriate buffer into your 1.5 microcentrifuge tube containing your ultra pure water.
  20. Use your 10 microliter Pipet to transfer 5 microliters of your wild type plasmid DNA into your 1.5 microcentrifuge tube containing your ultra pure water and your appropriated buffer.
  21. Remove the container of the proper restriction enzyme from the -20 degrees Celsius refrigerator and store it in a cold box. The reason why enzymes must be kept in this container is due to the fact that they degrade extremely easily and also because they are expensive, and must be maintained in this stable condition.
  22. Use your 2.5 microliter Pipet to transfer 2 microliters of your restriction enzyme into your 1.5 microcentrifuge tube containing your ultra pure water, your appropriated buffer, and your MiniPrep mutation plasmid DNA.
  23. Use your 2.5 microliter Pipet to transfer 2 microliters of your restriction enzyme into your 1.5 microcentrifuge tube containing your ultra pure water, your appropriated buffer, and your wild type plasmid DNA.
  24. Place your 1.5 microcentrifuge tubes containing your ultra pure water, your appropriated buffer, your MiniPrep mutation plasmid DNA, and your ultra pure water, your appropriate buffer, and your wild type plasmid DNA respectively into the incubator with the proper incubation temperature for your restriction enzyme for an hour.
  25. During the time period wait, make sure to prepare 2 0.6 microcentrifuge tubes for each mutation, one for the mutated plasmid DNA, and the other for the wild type plasmid DNA. Label them accordingly with your name/initials, the date, the name of your mutation or the name of your wild type plasmid DNA, and the step at which you are currently at within your experiment.
  26. Use your 10 microliter Pipet to transfer 5 microliters of ultra pure water into each 0.6 microcentrifuge tube.
  27. Use your 10 microliter Pipet to transfer 3 microliters of the loading dye into each 0.6 microcentrifuge tube.
  28. Use your 10 microliter Pipet to transfer 2 microliters of the mutated plasmid DNA into each respective 0.6 centrifuge tube as well as 2 microliters of the wild type plasmid DNA into each respective 0.6 centrifuge tube.
  29. Take a diagnostic gel with small wells and place it into a 1X TAE buffer in the gel electrophoresis box.
  30. Use your 10 microliter Pipet to transfer 10 microliters of the Ladder into the side well of the diagnostic gel.
  31. Use your 10 microliter Pipet to transfer 10 microliters of the ultra pure water, dye, and mutated plasmid DNA mixture as well as your 10 microliters of the ultra pure water, dye, and wild type plasmid DNA mixture to load into the small wells of the diagnostic gel. Remember to pipet up and down in order to blend evenly before you load into the small wells of the diagnostic gel.
  32. Put on the lid of the gel electrophoresis box, and run for approximately 15-20 minutes. Check in 5 minute intervals to check whether or not the DNA has run toward the third line of the boat tray that holds the gel.
  33. During gel electrophoresis, open the Carestream Gel Logic Model 112 machine, and place saran wrap on its surface. Make sure that the saran wrap is evenly spread out and that there are no air bubbles and creases-- these disturbances may interfere with the photograph of your gel.
  34. After gel electrophoresis, place your gel into the Carestream Gel Logic Model 112 machine and lose the door. Turn on the ultraviolet light, and open the Carestream MI Application on your computer. Select the “Capture GL 112” option and then choose “Preview” and then select “Capture”. Invert the photograph and make sure the picture is unsaturated so that the lanes look a lot more clear.
  35. Store the picture into the image database, and the experiment “Restriction Enzyme Digestion Subsequent to MiniPrep” with the type as “Diagnostic Gel”. Edit the description so that the amount of time the gel ran is recorded as well as the lanes associated with the PCR products in addition to the location of the ladder.
  36. Print the photograph and tape it into your laboratory notebook. Label the lanes with the names of the PCR products as well as the ladder.
  37. Sanitize your lab bench, and remove your personal protective equipment.
  38. Wash your hands thoroughly.
 

Protocol: Purification after the Linearization of DNA
 

Objective: The purpose behind the purification after the linearization of DNA is to create a completely purified version of the linearized mutation plasmid DNA so that it is completely ready for the in vitro RNA transcription-- the last and final step of the mutagenesis protocol. It is also an extremely important aspect to the experiment.
 

Materials:
 
  • Restriction Enzymes
  • Buffered saturated phenol solution
  • Chloroform
  • 3M Sodium Acetate (NaAc) pH 5.5
  • Ethanol (100% and 70%)
  • DNase, RNase free water
  • 1.5 microcentrifuge tubes
Procedure:
 
  1. Bring the buffered saturated phenol (in fridge) to room temp and gather the necessary ingredients:  chloroform (w/ IsoAmyl Alcohol), 3M NaAc and EtOH.  Note: you can use chloroform without Isoamyl Alcohol, but we usually use a ratio of 24:1 (chloroform : Isoamyl Alcohol).  
  2. Add an equal volume of buffered saturated phenol to the tube containing the linearize shuttle plasmid and shake vigorously for about 3 min.
  3. Centrifuge at 14,000 rpm in the microfuge for 5 minutes.
  4. Transfer the upper layer to a new microfuge tube (try not to get any of the phenol).  Discard phenol.
  5. Add an equal volume of chloroform to the sample and shake vigorously.
  6. Centrifuge at 14,000 rpm in the microfuge for 5 minutes.
  7. Transfer the upper (aqueous) layer to a new tube (try not to get any of the chloroform).  Discard chloroform.
  8. Add a 1/10 volume of 3M NaAc pH 5.5 to the sample (i.e., if you have 500 µl, then add 50 µl NaAc).
  9. Add 2X volume of 100% EtOH (i.e., 1.0 ml EtOH to 500 µl volume).
  10. Mix and place at -20°C for 30 minutes
  11. Pellet sample in microfuge at 14,000 rpm for 10 minutes.
  12. Check to see that there is a pellet.
  13. Remove the supernatant after you see a pellet.  Rinse with 1 ml of 70% EtOH and quick spin for 3 min at 14,000.  Remove the sup after you see a pellet.  Let it air dry.
  14. Resuspend the pellet in DNAse, RNAse-free H2O.  
 

Protocol: In Vitro Transcription of RNA
 

Objective: The final step of the mutagenesis protocol is to transcribe RNA from the linearized DNA we generated from the previous portion. This will provide the electrophysiology unit of the Cui Lab with the type of mutated RNA they necessitate in in order to inject the mutated cells into the frog’s oocytes and to detect changes within the function and the shape of their membranes’ ion channels.
 

Materials:
  • 40 microliters Linearized DNA Mixture
  • 3 microliters Appropriate Restriction Enzyme
  • 2 microliters Ultra Pure Water
  • 5 microliters 10X BCS Buffer
  • 200 microliters Pipet
  • 10 microliters Pipet
  • 1.5 microcentrifuge tube for each Linearized DNA Mixture
  • Incubator
 

Procedure:
 
  1. Put on your personal protective equipment, such as your laboratory coat, in addition to your gloves, your facial protective gear, i.e. lab goggles.
  2. Prepare a 1.5 microcentrifuge tube for each linearized DNA mixture you have under your possession.
  3. Using a 10 microliter Pipet, transfer 2 microliters of the nuclease-free water into your 1.5 microcentrifuge tube.
  4. Using a 10 microliter Pipet, transfer 5 microliters of the 10X BCS buffer into your 1.5 microcentrifuge tube.
  5. Using a 200 microliter Pipet, transfer 40 microliters of the linearized DNA mixture into your microcentrifuge tube.
  6. Remove the container of the proper restriction enzyme from the -20 degrees Celsius refrigerator and store it in a cold box. The reason why enzymes must be kept in this container is due to the fact that they degrade extremely easily and also because they are expensive, and must be maintained in this stable condition.
  7. Using a 10 microliter Pipet, transfer 3 microliters of the appropriate enzyme into the 1.5 microcentrifuge tube.
  8. Incubate the in vitro RNA transcription reaction for 1 or 2 hours in the proper inubation temperature for the restriction enzyme.
  9. Afterwards, the RNA is ready to be utilized throughout electrophysiology in order to inject into the membranes of the frogs’ oocytes to analyze changes/fluctuations within their ion channels.
 

Protocol: Restriction Digestion for the Linearization of DNA
 

Objective: The purpose behind the linearization of the DNA is basically to prepare the linear, single-stranded DNA in order to induce the in vitro transcription RNA. It will also remove excess debris, such as enzymes, buffers, etc. This DNA linearization process is an important component to the success of the further progress of the mutagenesis protocol.
 

Materials:
 

10X NEB Buffer
BSA 100X
Ultra Pure Water
NEB NotI (10 U / uL)
Phenol/Chloroform/Isoamyl Alcohol Mixture (25:24:1)
7.5 M NH4OAc
100% Isopropanol
Gauge Needle
Heat Block
Centrifuge
Vortex
Laboratory Coat
Gloves
2.5 microliter Pipet
10 microliter Pipet
20 microliter Pipet
Incubator
Ladder
Protective facial gear, i.e. Lab Goggles
2 1.5 Microcentrifuge Tubes for Each Mutation
2 0.6 Microcentrifuge Tubes for Each Mutation
6 microliters dye
 

Procedure:
  1. Make Linearization Master Mix:
           per reaction:
           10 uL               10 X NEB Buffer #3
           1 uL                 BSA 100 X
           61 uL               DI H2O
           3 uL                 NEB Not1 (10 U / uL)
           ---------------------------------------------
           75 uL
  1. In a sterile 1.5 mL microcentrifuge tube, add 75 uL of Linearization Master Mix to 25 uL of mini prep plasmid DNA (5 – 10 ug).  Mix by pipetting.
  2. Incubate at 37C overnight (> 14 hr).
  3. Run a few representative samples on a 1 % agarose gel (ex: 0.5 g agarose + 1 mL 50 X TAE buffer + ~49 mL DI H2O) to verify linearization.  (each sample – 1 uL reaction + 9 uL DI H2O + 1.7 uL 10 X Sample buffer) (Run at 170 V for 5 minutes against a 1 kb ladder)
  4. Add 100 uL of phenol / chloroform / isoamyl alcohol (25:24:1) to each tube.  Vortex for 30 seconds and then spin at high speed for 5 minutes.  Transfer 95 uL of the aqueous phase (top layer) into a sterile 1.5 mL tube.  Make sure no interface or organic phase is removed.
  5. Add 100 uL of chloroform / isoamyl alcohol (24:1) to each tube.  Vortex for 30 seconds and then spin at high speed for 5 minutes.  Transfer 95 uL of the aqueous phase (top layer) into a sterile 1.5 mLtube.  Make sure no interface or organic phase is removed.
  6. Add 30 uL of 7.5 M NH4OAc (Ammonium acetate) to aqueous layer.
  7. Add 130 uL of cold (4C) 100 % isopropanol.  Vortex and place at -20C for > 30 min.
  8. Spin tubes at high speed (4C) for > 30 min.  Aspirate the supernatant with a 27 gauge needle, being careful not to aspirate the DNA pellet.
  9. Gently add 500 uL of cold (-20C) 80 % ethanol to DNA pellet.  Spin for 10 min. at 4C.  Aspirate the supernatant with a 27 gauge needle.
  10. Dry in a 37 C heat block for 15 – 20 min.  Verify that all the EtOH has evaporated.
  11. Resuspend dried pellet in 10 uL of sterile RNase free DI H2O and store at -20C.
  12. During the time period wait, make sure to prepare 2 0.6 microcentrifuge tubes for each mutation, one for the mutated plasmid DNA, and the other for the wild type plasmid DNA. Label them accordingly with your name/initials, the date, the name of your mutation or the name of your wild type plasmid DNA, and the step at which you are currently at within your experiment.
 
  1. Use your 10 microliter Pipet to transfer 5 microliters of ultra pure water into each 0.6 microcentrifuge tube.
 
  1. Use your 10 microliter Pipet to transfer 3 microliters of the loading dye into each 0.6 microcentrifuge tube.
 
  1. Use your 10 microliter Pipet to transfer 2 microliters of the mutated plasmid DNA into each respective 0.6 centrifuge tube as well as 2 microliters of the wild type plasmid DNA into each respective 0.6 centrifuge tube.
 
  1. Take a diagnostic gel with small wells and place it into a 1X TAE buffer in the gel electrophoresis box.
 
  1. Use your 10 microliter Pipet to transfer 10 microliters of the Ladder into the side well of the diagnostic gel.
 
  1. Use your 10 microliter Pipet to transfer 10 microliters of the ultra pure water, dye, and mutated plasmid DNA mixture as well as your 10 microliters of the ultra pure water, dye, and wild type plasmid DNA mixture to load into the small wells of the diagnostic gel. Remember to pipet up and down in order to blend evenly before you load into the small wells of the diagnostic gel.
 
  1. Put on the lid of the gel electrophoresis box, and run for approximately 15-20 minutes. Check in 5 minute intervals to check whether or not the DNA has run toward the third line of the boat tray that holds the gel.
 
  1. During gel electrophoresis, open the Carestream Gel Logic Model 112 machine, and place saran wrap on its surface. Make sure that the saran wrap is evenly spread out and that there are no air bubbles and creases-- these disturbances may interfere with the photograph of your gel.
 
  1. After gel electrophoresis, place your gel into the Carestream Gel Logic Model 112 machine and lose the door. Turn on the ultraviolet light, and open the Carestream MI Application on your computer. Select the “Capture GL 112” option and then choose “Preview” and then select “Capture”. Invert the photograph and make sure the picture is unsaturated so that the lanes look a lot more clear.
 
  1. Store the picture into the image database, and the experiment “Restriction Enzyme Digestion Subsequent to” with the type as “Diagnostic Gel”. Edit the description so that the amount of time the gel ran is recorded as well as the lanes associated with the PCR products in addition to the location of the ladder.
  2. Print the photograph and tape it into your laboratory notebook. Label the lanes with the names of the PCR products as well as the ladder.
 
  1. Check to see if the mutations are positive. If they all are, which they should be, begin preparing for the purification of the linearized product using the Promega kit.
 
  1. Sanitize your lab bench, and remove your personal protective equipment.
 
  1. Wash your hands thoroughly.
 

Protocol: Making 5X TBE (10X Running) Buffer (1L)
 

Tris base (Fisher, BP 152-500) 54 g
Boric Acid (Fisher BP 168-500) 27.5 g
0.5 M EDTA (pH 8.0) 2 mL
dH2O 1 L
 

Protocol: Making 50X TAE Buffer (1L)
 

Tris base (Fisher, BP 152-500) 242 g
Glacial acetic acid 57.1 mL
0.5 M EDTA (pH 8.0) 100 mL
dH2O 1 L
 

TELT Buffer (500 mL)
 

Tris.Cl (pH 7.5) 25 mL  1 M Tris.Cl (pH 8.0)
62.5 mM EDTA 62.5 mL 0.5 M EDTA (Or 312.5 mL 0.1 M EDTA)
25 M LiCl 53 g
0.4% Triton X-100 2 mL 100% Triton X-100
ddH2O To 500 mL
 

Protocol: Refilling Tips / Tubes
 

Procedure:
 
  1. Remove bag of new tips/tubes from the cabinet across from the 4 degrees Celsius refrigerator.
  2. Place into the original container appropriately.
  3. Cover the original container with a piece of laboratory tape and a piece of autoclave tape.
  4. Place the original container with the new tips/tubes into the autoclave bin. It should be autoclaved and sterilized within several days.
 

Protocol: Making LB Medium
 

Making LB Medium (1000 mL)
 
  1. 10 g Bacto-typton (Difco )
  2. 5 g Bacto-yeast extract (Difco )
  3. 10 g NaCl (Fisher 271-3 )
  4. 950 mL ddH2O
 

Mix and add 1.0 mL 1N NaOH
 

pH = 7.0
 

Fill to 1000 mL with dd H2O
 

Autoclave
 

If necessary, antibiotics should be added right before the culture.
(Ampicillin at 50 ug/mL) [Dilute 1:1000, 50 mg/mL stock)
 

Protocol: Making LB Plates
 

Making Agarose Plate (1000 mL)
 
  1. Make 1000 mL LB medium as above.
  2. Pour the medium into 2 flasks (500 mL each).
  3. Add 7.5g agar (Difco) in each flask (There is no need to stir).
  4. Autoclave and swirl the bottle to mix the agar.
  5. Cool to approximately 50 degrees Celsius. (If needed, add ampicillin to final concentration of 50 ug/mL).
  6. Pour plates (About 30 mL in each 100 mm plate, avoid air bubbles on the plate).
 

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