QuikChange Mutagenesis Protocol 1) Make sure template to be QC'd is in TOPO II (KanR) or bacterial vector (pUC57, AmpR). 2) Optional: Include promoter and/or ADH1(term). Allows for a very fast Not1/SpeI subclone over to yeast vectors (e.g. pRS315 or pRS316). 3) Design primers such that there is at least 15-20 bps of flanking primer sequence on either side of the mutation(s). [Note: QC can be used to mutate 1,2, or 3 changes at once, spanning several bps distance. For more than 3 base changes at once, use successive rounds and primer sets. For mutations that are too far apart (e.g. 25 bps apart), use successive rounds of QC primers.] 4) Use Stratagene's QuikChange Tm calcµLator and aim for a Tm of at least 79-80C, ideally 8083C (using the calcµLator). [81.5+0.41*(%GC(Integer)-675/(primer length)-N] where N= number of bases to be mutated/total primer length x 100. 5) Also create the rev. comp. (reverse) primer. 6) Dilute primers to 100 pmol/µL with water. Wording dilution is 1:10 dilution in water (10 pmol/µL final volume). 7) Set up Quickchange PCR reaction with KOD hot start polymerase (or other PCR reaction). Make sure to thaw all components and vortex to mix thoroughly. Precipitate can sometimes form upon arrival on dry ice. 3 µL Mg2+ 5 µL of dNTP mixture 5 µL of KOD 10x buffer 5 µL of F primer (working dilution) 5 µL of R primer (working dilution) 25.5 µL water (pure from Smart2pure system class I water) 1 µL of DNA template (purified miniprep DNA) diluted 1:10 in water or EB buffer 1.0 µL KOD enzyme (add last) Final volume should be 50 µL. [Can prepare PCR at room temp, but keep KOD enzyme in cold rack/ice until needed.] 8) Prepare KOD PCR program on thermocycler. 1. 95°C 2 minutes hot start 2. 95°C 15 seconds denature 3. 50.5°C to 53.5°C anneal temp (test experimentally) for 30 seconds 4. 72°C extension temp for 35-40 seconds per 1000 bps of product (include entire vector + insert in size calcµLation) 5. Go to Step 2 x 15 additional times (16 total cycles) 6. 72°C for 10 minutes 7. End program, hold at 15°C forever, can store in fridge at 4°C. 8) Run out 5 µL of PCR product on a DNA gel to make correct product size is present. 9) Set up Dpn1 digest (50 µL): with 5 µL of CutSmart buffer (thawed and vortexed), 0.5 to 1.0 µL of enzyme, 8 µL of the PCR reaction, and 36.5 µL water. 10) Run for 3 or more hours at 37°C (or overnight) and then 80 degrees for 20 min to stop reaction (optional stop at 80C). 11) Transform Competent TOP10 E. coli (1/3 tube each) with 2-3 µL of the Dpn1-treated reaction into 60-80 µL of bacterial cells. 12) Be sure to split the final bacteria + SOC (or LB broth) volume over a few plates (say, 1/10 volume on 1, 1/20 on another, then a bunch on the last). Recommended to only plate roughly 40-80 µL of the final bacteria + SOC/LB mixture on agar plate and spread for single colonies with sterile spreader. Incubate overnight at 37°C with plate upside down. 13) Miniprep 1 isolate and sent to DNA sequencing to confirm. If incorrect, send second and/or third isolates.