PCR

NB Cross-check the instructions given below with those of the manufacturer for the particular enzyme you are using.

0.5 ul template DNA

5ul buffer (usually containing tris, MgCl2, NaCl)

2.5ul dNTP stock (ie ATP, TTP, CTP and GTP). Stock concentration is 2.5mM.

2.5ul of each primer, to a final concentration of 1uM

1ul of DNA polymerase (eg Phusion)

36ul H2O

Keep the dNTP stock on ice at all times. Mix the above in a thin-walled PCR tube, adding enzyme just before placing in the PCR machine.

Site-directed mutagenesis (Quikchange)

The PCR product is an unmethylated 'open' plasmid (ie not a complete circle). The closed (WT) template plasmid is most efficient at transformation back into bacteria and so the original template must be degraded. Since this is normal methylated DNA from bacteria it can be degraded with the enzyme DpnI (which cuts the 2nd half of the BamHI restriction site only if methylation is present).

Add 1ul of DpnI to each sample and incubate at 37oC for 1hr.

Things to try if this isn't working for you:

(in no particular order)

  • Do a gradinet PCR to check annealing temperature

  • Use PfuUltra for quikchange PCR

  • Use KOD for inverse PCR (outward PCR) - tricky when template >8-10kbp. Don't use KOD for quikchange as you get lots of random inserts.

  • Template concentration is the thing that seems to help/hinder PCR most so try different concentrations

  • Try using 'standard' annealing temperatures of eg 50/55/60. As a very rough rule of thumb try 50% GC content at 50oC, 60% at 55oC and anything over 70% at 60oC

  • Make sure that your polymerase is non-strand displacing (ie don't use a mixture that contains Taq and also, if using Pfu/Pfx you're supposed to keep the extension temp at 68oC not 72oC)