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From 2012.igem.org

Kunkel Mutagenesis

Contents 1 Kunkel Mutagenesis 1.1 Produce ssDNA (RECOMMENDED TO DO IN DUPLICATE! SEE NOTE AT END OF SECTION 2) 1.2 Harvesting ssDNA 1.3 Kinasing Oligos 1.4 Anneal Diluted Oligo and ssDNA 1.5 Polymerize DNA 1.6 Transform 1.6.1 Electroporation Method 1.6.2 Heat Shock Method 1.7 Buffers

Produce ssDNA (RECOMMENDED TO DO IN DUPLICATE! SEE NOTE AT END OF SECTION 2)

• Transform plasmid into chemically competent CJ236 cells
  • Plate onto a Chlor+YOUR ANTIBIOTIC plate and incubate overnight at 37°C
• Inoculate 6 colonies into 3ml of LB + YOUR antibiotic (NO chlor here per NEB instructions)
• Grow for 4-6 hours at 37°C, shaking at 200rpm (until cloudy)
• Add 3ul of M13K07 helper phage
• Continue growing for 1 hour at 37°C, shaking at 200rpm
• Expand culture by diluting 1mL into 50ml of LB + antibiotic in 250ml flask
• Grow for 12 hours (no more than 16) at 37°C, shaking at 200rpm

Harvesting ssDNA

• Spin down overnight culture in sterile 50ml Falcon tube at 7000rpm for 20 minutes at 4°C
• Transfer supernatant (contains phage) to a new sterile 50ml Falcon tube
• Add 10ml 20% PEG/2.5M NaCl and mix thoroughly
• Incubate on ice for 45 minutes
• Spin down the phage at 7000rpm for 20 minutes at 4°C
• Decant liquid and let tube stand upright to drain off the rest of the liquid
• Resuspend the pellet in 1mL 1xPBS (Vortexing is okay)
• Transfer the 1mL into a microfuge tube
• Spin at 14,000rpm for 5 minutes
• Transfer the supernatant to a new microfuge tube with 300ul PEG/NaCl
• Vortex and incubate at room temperature for 10 minutes
• Spin down phage at 14,000rpm for 2 minutes
• Pipette off supernatant; do a second quick spin to collect residual liquid and pipette it off
• Resuspend the pellet (phage) in 1ml 1xPBS
• Spin down at 14,000rpm for 5 minutes
• Transfer the supernatant (phage) to a new microfuge tube
• Harvesting ssDNA from Phage using Qiagen Qiaprep M13 kit (#27704)
  • DO NOT SPIN OVER 8000rpm when using this kit!
  • Final solution should be greater than 20ng/uL. If not, try again; ssDNA can randomly fail, so it is best to make 2 batches in parallel (works as a nice counter balance as well).

Kinasing Oligos

• Design mutagenic oligo using Stratagene’s primer design and order the 5’->3’ antisense oligo
  • This is specific for pET29b+, other vectors may require the sense oligo
  • [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&SubPageType=ToolQCPD&PageID=15| QuikChange Primer Design Program]
  • Login Link
• Make Kinase Reaction Mix (make #rxn+2)
  • 3uL Kinase Buffer/rxn
  • 1uL of 10mM ATP/rxn
  • 1uL T4 Polynucleotide Kinase/rxn
  • 18uL ddiH2O/rxn
• Combine oligo and reaction mix in PCR strip tubes or 96well PCR Plate
  • Aliquot 23uL of Kinase Reaction Mix using repeater
  • Make sure all liquid at the bottom by tapping or briefly spinning
  • Add 7uL of 100uM oligo to the BOTTOM of each well and pipette up and down
• Seal, mix gently by tapping, and incubate at 37deg C (metal bath or PCR machine) for 1 hour
• Store on ice short term, or -20 long term
  • These can be re-used in the future so no need to throw them away!

Dilute Mutagenic Kinased Oligo (Optimal molar ratio is 1:4 dU-ssDNA:Oligo)

• Add 2uL of oligo into 200uL of diH2O
  • If doing a mutant with multiple oligos add 2uL of EACH oligo to the same tube
  • This dilution factor will achieve the desired 1:4 dU-ssDNA:Oligo molar ratio for most single stranded DNA preps.

Anneal Diluted Oligo and ssDNA

• Combine 0.2uL of T4 DNA Ligase Buffer with 2uL of ssDNA, (make #rxn+2)
• Aliquot 2.2uL of the mix into a in a fresh PCR plate or strip tubes
  • Make sure all the liquid as the bottom by spinning down or tapping
• 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
  • 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.
• Seal, mix (tapping or plate mixer)
• Run Siegel: Anneal in PCR machine (USE heated lid)
  • Starts at 95 degrees and ramps down to 25 over an hour
  • I find the slow ramp decreases background and increases overall kunkel efficiency relative to the quick protocol, which does 95-2min; 50-2min; 25-forever

Polymerize DNA

• Make polymerization reaction mixture (make #rxn+2)
  • 0.6uL 10x T4 Ligase Buffer/rxn
  • 0.4uL 25mM dNTPs/rxn
  • 0.4uL 10mM ATP/rxn
  • 0.4uL T7 Polymerase (unmodified from NEB)/rxn
  • 0.4uL T4 Ligase/rxn
• Add 2.2uL of polymerization reaction mixture to each annealing reaction
• Seal, mix (tapping or plate mixer)
• Incubate at room temperature for 1+ hrs.

Transform

Electroporation Method

• Dialyze DNA (ORDER IS IMPORTANT HERE!!!!):
  • Add ~40mL of diH2O to an empty Petri dish (till it is mostly full)
  • Mark up to 8 spots on the shiny side of a 0.025um membrane filter
  • Place the filter into the diH2O so shiny side is up and it is flat
    • DO NOT SUBMERGE!
  • CAREFULLY pipette the 5uL reaction onto the designated spots
  • Let dialyze for 45+ minutes
  • While DNA is dialyzing, remove an aliquot of BL21 DE3* electro-competent cells from Box labeled “Jasmine”, tubes labeled E*)
  • Place the cell aliquots on ice and allow them to thaw.
  • Aliquot 40 uL of each aliquot into a separate, labeled, sterile Eppendorf tube.
• Electroporate:
  • CAREFULLY remove as much of the DNA as possible from the membrane and pipette into an Eppendorf tube containing 40 uL of thawed electro-competent BL21 DE3* cells.
  • Transfer cell/DNA mixture to a 0.1cm electroporation cuvette which has been pre-chilled on ice, and return the cuvette to the ice.
  • Place into BioRad electroporater (only fit in 1 way) set to Bacteria and hit “Pulse”
  • IMMEDIATELY add 300uL of media (TB or LB, NO antibiotic) into the cuvette to extract cells, transfer to a fresh strip tube or PCR plate.
• Recover and Plate:
  • Incubate transformed cells at 37°C for ~30-45 minutes
  • Plate out ~200uL of each reaction on individual plates
  • Add ~10 glass beads/plate
  • Shake to spread
  • Turn plate and tap so beads are transferred to the lid, than pour out beads into 70% EtOH filled beaker.
• Turn plates so agar side is up and incubate at 37°C overnight

Heat Shock Method

• In a 14mL falcon add 5uL of Polymerization reaction and 50uL of BL21(DE3)* (Chemical comp NOT electro comp)
• Incubate on ice for 20+ minutes
• Heat Shock by submerging the bottom of the tube in a 42deg water bath for 1 minute
  • 45 seconds to 90 seconds is fine, 1 minute is generally safe
• Return to ice for ~2minutes
• Add 200uL of TB no antibiotic
• Recover by shaking at 37deg for 1hr
  • On individual plates plate out ~200uL of each reaction
    • I find it works best to do 12 plates at a time
  • Add ~10 glass beads/plate
  • Shake to spread
  • Turn plate and tap so beads are transferred to the lid, than pour out beads into 70% EtOH filled beaker.
  • Move to the next row.
• Turn plates so agar side is up and incubate at 37deg overnight

Buffers

20%PEG/2.5M NaCl

• 200g PEG (polyethylene glycol) 8000MW
• 141.6g NaCl
• Add dH2O to 1L
• Autoclave with a stir bar, stir immediately after autoclaving until cool, and store at 40C.

1xPhosphate Buffered Saline (PBS)

• 800ml dH2O
• 8g NaCl
• 0.2g KCl
• 1.44g Na2HPO4
• 0.24g KH2PO4
• Adjust pH to 7.4
• Add diH2O to 1L
• Autoclave