Rao:Mega-Primer

Mega-primer PCR is a polymerase chain reaction based method to introduce point mutations at internal locations in a designated gene or DNA template through two (or more) rounds of PCR using multiple forward or reverse priming oligonucleotides. Please see a standard cloning manual for a more in depth description.

=Materials=
 * High fidelity polymerase (Phusion, Pfu Turbo, and on and on)
 * 10 mM PCR grade dNTPs
 * Purified template (chromosomal or plasmid DNA)
 * Forward / Reverse DNA oligonucleotide primers (typically 3 primers are needed)
 * ddH2O

=Primer Design= Depending on the location of the desired mutation it can be benificial to either use an initial forward or reverse oligonucleotide primer to introduce the desired point mutation. In general if: So after deciding this you should have ordered:
 * the locus of mutation is towards the "beginning" of the gene, use an initial reverse primer to introduce the mutation
 * the locus of mutation is towards the "end" of the gene, use an initial forward primer to introduce the mutation
 * One forward primer to amplify your region of DNA (Primer 1)
 * One reverse primer to amplify your region of DNA (Primer 2)
 * One forward/reverse primer bearing your mutation(s) (Primer 3)

=PCR Round 1= This is the step in which you will be amplifying your "Megaprimer".

A standard 50 uL PCR protocol might be:
 * x uL of polymerase buffer
 * 1 uL template 5-10x diluted
 * 0.5 uL Primer 1 (or 2) (30 uM)
 * 0.5 uL Primer 3 (30 uM)
 * 1.5 uL MgCl2 (50 mM)
 * 1.5 uL DMSO (optional)
 * 1 uL dNTPs
 * x ddH20

Use the manufacturer's instructions to use your polymerase. Do only 20 to 25 cycles of amplification. Purify this PCR product by standard methods (Qiagen PCR prep or EtOH precipitation).

=PCR Round 2= This the step where you use your "Megaprimer" and amplify your entire region with it. Get why its called megaprimer now?

A standard 50 uL PCR protocol might be:
 * x uL of polymerase buffer
 * 1 uL template 5-10x diluted
 * 0.5 uL Primer 2 (or 1) (30 uM)
 * 5 uL Primer 3
 * 1.5 uL MgCl2 (50 mM)
 * 1.5 uL DMSO (optional, often necessary here)
 * 1 uL dNTPs
 * x ddH20

Amplify using manufacturer's instructions. You might need to vary the number of cycles (I typically use 25 here). Also, as the annealing temperature can be difficult to optimize for long products, other methods such as touchdown are very useful.

Purification of this product should be done by standard gel purification as your megaprimer is typically too large to be purified out using standard methods (Qiagen PCR prep or EtOH precipitation).

Now just digest and ligate into your favorite vector. Voila you should have your mutated region of DNA.