Introduction

This protocol was inspired (after failed site directed mutagenesis attempts using Quikchange) by the protocol listed here at the Colgate website here. There are several other method papers that describe this (or variant) MEGAPRIMER and MEGAWHOP protocols, there is one here and the original here.

This technique is useful for introducing single base pair, single amino acid, or multiple changes into a gene residing on a plasmid that you already have. In short, one mutagenic primer is paired with a standard primer to create a “Megaprimer” product that contains your mutation(s) of choice, is 200-400 bp long, and contains at least >=15 bp of homology on each end to your template plasmid. (You could, of course, just order a gene block with of this size containing all of your desired mutations and use it as a megaprimer). This megaprimer is purified and then used as primer with the template plasmid in a second PCR whole plasmid (Megawhop) reaction. This PCR product is cleaned up and transformed into competent cells to yield your desired mutation.

Required Materials

Template Plasmid
Competent Cells
PCR Reagents
Culturing Materials (>= 4 plates with appropriate antibiotics for transformations)
Sequence of desired mutation
Primer Upstream of Desired mutation

Procedure

1. Design a mutagenic primer with your desired mutation. Ensure at least 15 base pairs of homology downstream of your mutation.
2. Create your megaprimer by running PCR using your upstream primer and designed primer with a standard Phusion protocol. Your template is your unmutated gene product. 2x 50 uL reactions are sufficient. This megaprimer will contain your desired mutations.

These steps have worked:

• Denature at 98 deg for 1 min.
• 30 cycles of:
  • 98 deg for 30 sec
  • 62 deg for 30 sec
  • 72 deg for 15 sec
• 72 deg for 5 min

3. Confirm your PCR was successful with a DNA agarose gel and gel purify your 200-400bp product. Elute with a small volume (20 uL).
4. Use this purified megaprimer product and your template plasmid in a 2nd PCR (2x 50 uL reactions). Your control in this reaction is a reaction without your megaprimer from the previous step. Use commercial Phusion for this reaction (or other super high fidelity polymerase). Each tube should contain very little template plasmid (1 uL or 50 ng) and 5 uL of your purified megaprimer product. Concentrations for the rest of your PCR reagents are standard.
These steps have worked:

• Denature at 98 deg for 2 min
• 30 cycles of:
  • 98 deg for 1 min
  • 62 deg for 30 sec
  • 72 deg for 3 min (based on a plasmid size of 5kb, you should adjust accordingly)
• 72 deg for 5 min

5. Add 1 uL DpnI to each tube to digest your template plasmid.
6. Optional You can run 5uL of your completed PCR reaction on an agarose gel and compare it to (1) your control reaction and (2)an unreacted mixture of the PCR components. You should see a faint band at your expected plasmid size only in your primer-containing reaction (DpnI should digest the template in both reactions).
7. PCR Cleanup both your sample and control reactions.
8. Transform 1-2uL of each reaction into competent cells using standard electroporation or chemical transformation protocols.
9. Plate dilutions of both reactions on appropriate antibiotic plates for selection.
10. If your sample shows more colonies than your control, the process was likely successful. Pick 3 clones to make stocks and send for sequencing (link)

-- Main.SeanLeonard - 05 Sep 2014