Kunkel Mutagenesis

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

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).

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.

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

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
Electroporate
- CAREFULLY remove the reaction and pipette into the bottom of the crevice in the 0.1cm electroporation cuvettes and KEEP ON ICE
- Add 30uL of electro competent BL21(DE3)* cells (Competent Cell Rack, Box labeled “Jasmine”, tubes labeled E*)
- 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 37C for ~1hr+
- On individual plates 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

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

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)