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QuikChange (Stratagene)This is a quick and reliable (and dead easy too!) method for PCR but costs more. It is, however, highly recommended if you need to make only a small number of mutants, or if you can't or won't do any molecular biology that's too complicated.
1) Synthesise 2 complementary oligonucleotides.(see notes)
2) Prepare reaction mixture:
5 ul of 10X reaction buffer
X ul of template DNA (5-50 ng)
X ul of each primer
1 ul of 10 mM dNTP mix (2.5 mM each dNTP)
make up to 50 ul with ddH2O
Add 1 ul of pfu DNA polymerase
3) Overlay reaction with mineral oil.
4) Start Cyling reaction.
cycle 1
95 degC for 30 sec.
cycle 2 use 12 to 18 cycles (see notes)
95 degC for 30 sec.
55 degC for 60 sec.
68 degC for 2 min/kb of plasmid length
5) Place on ice to chilled to <37 degC.
6) Add 1 ul of Dpn 1 restriction enzymes (10 U/ul) directly to each amplification reaction below the mineral oil.
7) Mixed reaction mixture gently by pipetting up and down. Centrifuge.
Incubate at 37 degC for 1 hour.
8) Use 1 ul of the reaction mixture to transform cells.
notes
1) primer design -
- Both oligos must contain the same mutation and anneal to same sequence.
- Mutation in middle with 10 - 15 bases complementary sequence on both side.
- length - 25-45 bases. Tm ~ 10 degC above extension temperature of 68 degC.
- GC content should be ~ 40% and primers should end in one or more C or G.
- Primers ideally should be FPLC or PAGE purified.
2) For point mutation - 12 cycles,
single aa change - 16 cycles,
multiple aa deletions or insertions - 18 cycles.
3) Be careful to avoid carrying over mineral oil when transforming cells - it can seriously reduce transformation efficiency.
4) If you fail to get colonies, try lowering the annealing temperature.
5) Keep primer concentration in excess, try varying template concentration.
6) For larger plasmid, this method may not work well. Optimisation may be necessary (e.g. increasing template and primer concentration, increasing extension and digest time, different polymerase etc.). Plasmid in excess of 10Kb may not work at all.
7) If the region to be mutated has a high GC content, the reaction may also be problematic
8) Digestion with Dpn1 to remove template DNA is an excellent idea that could be extended to other applications or mutagenesis protocols.
9) If you do not wish to pay for the kit, you only need to purchase the enzymes separately which will make this cheaper.
