Phusion mutagenesis protocol - version 2.0, Dec 2010.  Adapted from NEB Phusion mutagenesis kit manual with assistance from Glen Cho (Massachusetts General Hospital).

Purpose:  To introduce targeted mutations into a circular plasmid template and recover the mutated product.

Concept:  Using primers which meet, but do not overlap, at the 3’ end you will PCR the entire plasmid, introducing the mutations in the 5’ end of your primers.  One or both primers can carry mutations or insertions.  Deletions of any size can be made by leaving a gap between the 3’ ends of the primers.

Primer design:  Start by designing two primers close to your mutation site which meet at the 3’ end and are “normal” PCR primers (e.g. 18-22 bp in length, Tm 55-65°C).  Add the required mutations to the 5’ extensions of one or both primers (just as if you were adding restriction sites for cloning etc) – do not add any clamps, spacers or padding.  The sequence should be the exact sequence you want to get in the final plasmid. The constraints upon the 5’ extensions are quite loose, just as for normal PCR, but try to keep mispriming and hairpins to a minimum. 

For deletions, space “normal” PCR primers the correct distance apart and do a regular PCR.  The gap between the 3’ ends will be deleted.

For insertions there are two possible strategies.  You can add the bases at the 5’ end of one primer, or in the middle of a primer, with 10-15 exact matching bases on either side.

For this modified method you do not need phosphorylated oligos – you will add kinase to the ligation reaction following the PCR.

Tip: If you have repeats or strings of the same nucleotide (e.g. GGGG, or CCC) at the 5’ end of a primer, make sure to include the whole repeat string in the primer with a couple of extra bases 5’, or shorten the primer to miss out the repeat entirely.  If you have a primer ending during a repeat, your risk of frameshift is increased, especially if you have mismatches directly 3’ to this.

 

Reaction conditions: 50 µl reactions

10 pg of template DNA

0.5 µM of each primer

10 µl HF buffer

1 µl 10 mM dNTPs

0.5 µl Phusion enzyme

XX µl water (to 50 µl)


Cycling conditions:

98°C     2 minutes

| 98°C          30 seconds

25x   | 55-65°C     30 seconds (primer dependant temperature)

| 72°C           5-10 minutes (30s -1 min per kb)

72°C      10 minutes

 

Visualising successful PCR product:  Following the PCR reaction I run 5 µl on a gel to check for product.  I also do a control reaction with no polymerase to ensure that visible product is a result of PCR.  Usually there is no band in the control, and a strong band of the full length size in the reaction lane.  If you get a product which doesn’t run out of the well, then your primers were overlapping and you have made a huge concatemer (congrats, this is an exclusive club of which I am one of the founding members).

Dpn1 digestion: Since the majority of the DNA is PCR product you should be able to proceed directly to ligation.  However, to control for inefficient ligation and ensure mutagenic product is transformed I add 1µl of Dpn1 directly to the PCR reaction and incubate at 37°C for 30-60 minutes to digest template DNA.

 

Ligation:

10 µl water

4 µl of PCR mix (following Dpn1 digestion)

2 µl of NEB buffer 4

2 µl of 10x ligation additions (10 mM ATP, 100 mM DTT)

1 µl PNK T4 kinase

1 µl T4 ligase

Incubate at r.t. for 20 minutes.  Transform 3-5 µl into 30-40 µl competent cells.

As a control you can also transform the Dpn1-treated no polymerase reaction to quantify template background.

 

I have made many constructs this way and have sequenced over 60 clones, all but 2 were successfully mutated.  If there are concerns about introducing secondary mutations in other portions of the plasmid as a result of PCR, you can cut and paste the insert into a “fresh” vector backbone.