Team:UC Davis/Protein Engineering Kunkel

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UC Davis iGEM 2014

Design

Design

Build

Build

Test

Test

Kunkel Mutagenesis

  1. Produce ssDNA (RECOMNEDED TO DO IN TRIPLICATE! SEE NOTE AT END OF SECTION 2)
    1. Transform plasmid into chemically competent CJ236 cells (initial stock is free from NEB, make your own from there. Any standard chem comp method will work). -Plate onto a Chlor+YOUR ANTIBIOTIC plate and incubate overnight at 370C.
    2. Inoculate 6 colonies into 3ml of LB + YOUR antibiotic. (NO chlor here per NEB instructions)
    3. Grow for 4-6 hours at 370C, shaking at 200rpm (until cloudy).
    4. Add 3ul of M13K07 helper phage.
    5. Continue growing for 1 hour at 370C, shaking at 200rpm.
    6. Expand culture by diluting 1ml into 50ml of LB + antibiotic in 250ml flask.
    7. Grow overnight at 370C, shaking at 200rpm.
  2. Harvesting ssDNA
    1. Spin down overnight culture in sterile 50ml Falcon tube at 7000rpm for 20 minutes at 40C.
    2. Transfer supernatant (contains phage) to a new sterile 50ml Falcon tube.
    3. Add 10ml 20% PEG/2.5M NaCl and mix thoroughly and incubate on ice for 45 minutes.
    4. Spin down the phage at 7000rpm for 20 minutes at 40C.
    5. Decant liquid and let tube stand upright to drain off the rest of the liquid.
    6. Resuspend the pellet in 2ml 1xPBS. Vortexing is okay.
    7. Spin at 14,000rpm for 5 minutes.
    8. Transfer the supernatant to new microfuge tubes with 300ul PEG/NaCl.
    9. Vortex and incubate at room temperature for 10 minutes.
    10. Spin down phage at 14,000rpm for 2 minutes.
    11. Pipette off supernatant. Do a second quick spin to collect residual liquid and pipette it off.
    12. Resuspend the pellet (phage) in 1ml 1xPBS.
    13. Spin down at 14,000rpm for 5 minutes.
    14. Transfer the supernatant (phage) to a new microfuge tube.
    15. Harvesting ssDNA from Phage using Qiagen Qiaprep M13 kit (#27704)
      1. DO NOT SPIN OVER 8000rpm when using this kit!
      2. Final solution should be greater than 20ng/uL. If not try again; ssDNA can randomly fail so often it is best to make 2 batches in parallel (works as a nice counter balance as well).
  3. Kinase oligo in a 96 well plate
    1. Design mutagenic oligo using statagene’s primer design and order the 5’->3’ antisense oligo
      1. This is specific for pET29b+, other vectors may require the sense oligo
      2. http://www.stratagene.com/sdmdesigner/default.aspx
      3. Alternatively, have the oligo encode: <18bp compliment>-<18bp compliment)
      4. If you are using multiple oligos for several changes at once, make sure they do not overlap. You may need to make a longer/shorter oligo to encode all the mutations on one oligo (but this is expensive after 100bp) or break it into several oligos with shorter compliments.
    2. Make Kinase Reaction Mix (make #rxn+2)
      1. 3uL PNK Buffer/rxn
      2. 1uL of 10mM ATP/rxn
      3. 1uL T4 Polynucleotide Kinase/rxn
      4. 18uL ddiH2O/rxn
    3. Combine oligo and reaction mix in PCR strip tubes or 96well PCR Plate
      1. Aliquot 23uL of Kinase Reaction Mix using repeater
      2. Make sure all liquid at the bottom by tapping or briefly spinning
      3. Add 7uL of 100uM oligo to the BOTTOM of each well and pipette up and down
      4. Seal, MIX gently by tapping, and incubate at 37deg C (metal bath or PCR machine) for 1 hour
      5. Store on ice short term, or -20 long term (These can be re-used in the future so no need to throw them away!)
  4. Dilute Mutagenic Kinased Oligo (Optimal molar ratio is 1:4 dU-ssDNA:Oligo)
    1. Add 2uL of oligo into 200uL of diH20 and then MIX WELL!
      1. If doing a mutant with multiple oligos add 2uL of EACH oligo to the same tube
      2. This dilution factor will achieve the desired 1:4 dU-ssDNA:Oligo molar ratio for most single stranded DNA preps.
  5. Anneal Diluted Oligo and ssDNA
    1. Combine 0.2uL of T4 DNA Ligase Buffer with 2uL of ssDNA, (make #rxn+2)
    2. Aliquot 2.2uL of the mix into a in a fresh PCR plate or strip tubes
    3. Make sure all the liquid as the bottom by spinning down or tapping
    4. Add 2uL of THE DILUTED kinased primer (single or mixed) to generate desired mutant to the bottom of the plate and PIPPETE UP AND DOWN TO MIX.
    5. Always do a background as well where you have ssDNA with NO oligo (just add 2uL of water). This will allow to you to know if you mutations work and estimate your mutation efficiency so you can pick the appropriate number of colonies you need to screen in order to find the mutation desired.
    6. Seal, MIX (tapping or plate mixer)
    7. Anneal Oligo to DNA (USE heated lid)
      1. Slow Anneal in a PCR machine: Start at 95 degrees and ramp down to 25 over an hour
      2. Fast Anneal in PCR Machine: 95-2min; 50-2min; 25-4eva *I find the slow ramp decreases background and increases overall kunkel efficiency relative to the quick protocol, but Fast Anneal is better for generating libraries
  6. Polymerize DNA
    1. Make polymerization reaction mixture (make #rxn+2)
      1. 0.6uL 10x T4 Ligase Buffer/rxn
      2. 0.4uL 25mM dNTPs/rxn
      3. 0.4uL 10mM ATP/rxn
      4. 0.4uL T7 Polymerase (unmodified from NEB)/rxn
      5. 0.4uL T4 Ligase/rxn
    2. Add 2.2uL of polymerization reaction mixture to each annealing reaction
    3. Seal, mix (tapping or plate mixer)
    4. Incubate at room temperature for 1+ hrs.
  7. Transform (electroporation is more reliable for kunkels than chem comp, but if high efficiency chem comp cells should work as well)
    1. Dialyze DNA (ORDER IS IMPORTANT HERE!!!!)
      1. Add ~40mL of diH2O to an empty Petri dish (till it is mostly full)
      2. Mark up to 8 spots on the shiny side of a 0.025um membrane filter
      3. Place the filter into the diH2O so shiny side is up and it is flat DO NOT SUBMERGE!
      4. CAREFULLY pipette the 5uL reaction onto the designated spots
      5. Let dialyze for 20+ minutes
    2. Electroporate
      1. CAREFULLY remove the reaction and pipette into the bottom of the crevice in the 0.1cm electroporation cuvettes and KEEP ON ICE
      2. Add 30uL of electro competent BL21(DE3)* cells (Competent Cell Rack, Box labeled “BL21*-ELECTROCOMP”, tubes labeled E*)
      3. Place into BioRad electroporater (only fit in 1 way) set to Bacteria and hit “Pulse”
      4. IMMEDIATLEY add 200uL of media (TB or LB, NO antibiotic) into the cuvette to extract cells, transfer to a fresh strip tube or PCR plate.
    3. Recover and Plate
      1. Incubate 37deg for ~1hr
      2. On individual plates (not sure how to get around this) plate out ~200uL of each reaction, I find it works best to do 12 plates at a time
        1. Add ~10 glass beads/plate
        2. Add the 200uL of solution
        3. Shake to spread
        4. Turn plate and tap so beads are transferred to the lid, than pour out beads into 70% EtOH filled beaker.
        5. Move to the next row.
      3. Dry plates at 37deg for ½ hr, lid-side up.
      4. Turn plates so agar side is up and incubate at 37deg overnight

    Stock Solutions:
    20%PEG/2.5M NaCl
    200g PEG (polyethylene glycol) 8000MW
    141.6g NaCl
    Add dH20 to 1L
    Autoclave with a stir bar, stir immediately after autoclaving until cool, and store at 40C.

    1xPhosphate Buffered Saline (PBS)
    800ml dH20
    8g NaCl
    0.2g KCl
    1.44g Na2HPO4
    0.24g KH2PO4
    Adjust pH to 7.4
    Add dH2O to 1L
    Autoclave

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