Kit-Free Site Directed Mutagenesis Protocol

Molecular biologists are familiar with the QuikChange® Site-Directed Mutagenesis Kit that allows rapid intoduction of a point mutant into a plasmid/vector/mammalian expression construct. Briefly, the protocol first involves a thermocycling/PCR step with mutagenic primers, followed by a DpnI digestion step to digest the methylated parental/wild-type plasmid, and finally transformation into competent cells for nick repair.

 An animation showing the basics of site directed mutagenesis. Image source: http://commons.wikimedia.org/wiki/File:Site-Directed-Mutagenesis.gif

An animation showing the basics of site directed mutagenesis. Image source: http://commons.wikimedia.org/wiki/File:Site-Directed-Mutagenesis.gif

 

Most experts we’ve talked to still use this technique, but don’t see the point of an expensive kit. Instead they use their own protocols with inexpensive enzymes and reagents bought separately. One such protocol involves the following:

“QuickChange” Protocol for Site-Directed Mutagenesis

Step 1: Design mutagenesis oligos (Primer 1 and Primer 2) using the website PrimerGenesis

Step 2: Order high quality custom oligos from tebu-bio

Step 3: Set up this reaction:

Plasmid DNA (50ng/ul) 1  ul
10x Reaction Buffer (comes with polymerase) 5 ul
50 mM MgCl2 (comes with polymerase) 1 ul
dNTPs (10mM) 1 ul
DMSO 2.5 ul
Primer 1 (10uM) 1 ul
Primer 2 (10uM) 1 ul
AccuStar™ DNA polymerase* 1 ul
Water 36.5 ul
TOTAL 50 ul

* Due to inherent 3’-to-5’ exonuclease activity of this very high fidelity enzyme, the polymerase must be added last to the reaction in order to prevent primer damage.

Step 4: Run the following PCR Cycle in a thermocycler

95°C 5 min; 15 cycles of (95°C 30sec, 60°C 1 min, 68°C 15 min*), 68°C 10 min, 10°C forever.

*for large (>9kb) plasmids, increase the extension time to 20min.

Note: run a negative control reaction without polymerase enzyme.

Step 5: Add 1 ul of Dpn1 enzyme to PCR tubes and leave at 37C for 2 hours

Step 6: Prepare a 1% agarose gel with GreenView™ Plus DNA Gel Stain

Step 7: Load 10ul of completed reaction mixed with SafeGreen™ Loading Dye next to 1kb DNA ladder and run the gel at 150V for 10 min to determine if PCR was successful. You should not see a product for the ‘negative control’ tube that had no polymerase. Troublshooting: If PCR reaction yields no product, then do gradient for the annealing temperature. The DMSO is extremely important, so keep this in the reactions.

Step 8: Mix PCR reaction with 500ul of PB buffer and purify on column. Wash with PE buffer and elute with 50 ul of water. Use 10ul of for transformation of TransforMax™ EC100™ competent cells.

 

Interested in using this protocol to your lab? Here is a list of products you will need:

Item Name Catalog Number
2′-dNTP Set 040N-2505-25 
Accustar DNA Polymerase 218ME-0067-05 
Molecular Biology Grade Agarose 218EP-0010-05 
GreenView Plus DNA Gel Stain 217N101 
SafeGreen Loading Dye 217D012 
TransforMax EC100 Chemically Competent E. coli 035CC02810 
1kb DNA Ladder 217D001

 

 

 

Legal Notes: QuikChange® is a registered trademark of Stratagene.

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