QuikChange XL protocol – modified by ZSD from Stratagene (version
1/11/2001).
1. Read the Stratagene description of QuikChange XL before proceeding further. We have
recently found that the XL version of the QuikChange kit is VERY reliable and we favor
using this kit for ALL plasmids.
2. Preparation of template: Template is typically prepared using the QIAprep Spin Miniprep
Protocol. Following our local protocol will yield concentrations in excess of 100 nM/L of
DNA; the amount of the final product is proportional (as to the initial amount of template,
because the QuikChange protocol amplifies only the parent strands. You need more template
than for normal PCR.
3. Primer choice: Two complimentary primers carrying the desired mutations are required. The
QuikChange strategy relies on a linear amplification reaction, which copies large templates; it
is worth the effort because of the time it saves—when it works. The protocol is dependent on
the choice of primers. In principle, when you have single base changes primers 25-35 bp
ought to be sufficient. In our experience, sometimes longer primers, in excess of 50 bp, will
work better. This protocol describes how to deal with the difficulties of longer primers,
including those with multiple base changes an/or deletions/insertions.
As a rule, IF IN DOUBT, ADD ANOTHER 5 BASES ON. DO NOT SAVE ON PRIMERS.
You can probably make a longer primer work one way or another, but if it fails to prime your
reaction, there is no way around it. Standard purity is adequate.
4. Polymerase choice. Use Pfu-Turbo from the kit.
NB: When dealing with very long primers and complicated multiple mutations
proceed directly to step [9].
5. Mutagenesis: Prepare the reaction, in thin walled PCR tubes, as follows*:
18  of water
2.5  of Pfu reaction buffer
1.0  of the template
0.5  of primer #1, diluted to 100 ng/ concentration
0.5  of primer #2 diluted as above.
0.5  of 10mM dNTP mix (2.5mM each NTP)
1.5  of the QuikSolution from the kit
add 0.5  of Pfu polymerase
overlay with 20 of PCR mineral oil (if your PCR machine requires oil)
(*the 25 L volume use here reduces the cost of polymerase by 50%
compared to standard protocols)
For positive control use standard sequencing primers with your template – this
shows you the viability of Pfu.
6. Set the PCR parameters according to the QuikChange protocol::
Window
1
2
3
Cycles Temperature (C)
1
96
18
96
60
68
1
68
Time
60 sec
50 sec
50 sec
2 min/kb of template
7 min
7. 1 hour before completion of the PCR prepare 50 ml of a 0.8% agarose gel. When the
is still hot add 4  of ethidium bromide (use rubber gloves!). Run the gel at 70 mA
with a suitable (e.g 1 kb) ladder for control (0.5L), and 2.5L of your reaction,
diluted by 7.5 L of water and 2L of loading dye. You should see three bands:
barely visible supercoiled and unwound circular template and in between much
stronger linear PCR product. If template concentration is low you will essentially see
only the product. If you are satisfied, proceed directly to DpnI digestion (step [10]).
8. If you fail to detect PCR product, run the reaction again, this time in 1% DMSO (i.e.
add 1 L of a 1:4 dilution of DMSO to your reaction mixture, reduce water by the
same amount). DMSO may help in priming, but sometimes introduces spontaneous
mutations, so use it sparingly. Run an agarose gel, and if your reaction has worked,
proceed to step [10].
9. If you still fail to observe a product or if you are dealing with long primers, carry out
two independent extensions with two reactions, set up as in [5], but one for each of
the two primers. This follows the protocol given by Bruce Malcolm (Biotechniques
1999 Apr;26(4):680-2: Wang W, Malcolm BA, Two-stage PCR protocol allowing
introduction of multiple mutations, deletions and insertions using QuikChange SiteDirected Mutagenesis). Reduce the number of cycles to 10. At the end, withdraw one
mixture from under the oil and inject under the oil into the other; add another 0.5 Pfu
and run the standard 25-20 cycle PCR. Run an agarose gel again. You should have a
PCR product now.
10. Inject 0.5 L of DpnI under the oil; if you do step [7], it is enough to use 20 L from
that volume to minimize the use of DpnI. Incubate at 37 for 1-2 hours. This always
works, but if in doubt run another agarose gel; you should see a nice ladder generated
by specific cleavage of your template with DpnI (unless you had very little template).
11. Proceed to transformation.