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Site Directed Mutagenesis (QuickChange Method)
Cornell iGEM 2012 Protocol
Source: Dylan Webster (Adapted from QuickChange II XL Site-Directed Mutagenesis Kit Protocol)
http://www.chem.agilent.com/Library/usermanuals/Public/200521.pdf
http://www.neb.com/nebecomm/products/protocolProductE0553.asp
Mutagenic Primer Design:
Use the Agilent webpage to help you design mutagenic primers: www.agilent.com/genomics/qcpd
In general, primer pairs should anneal to the same sequence of opposite strands, contain the
desired mutation flanked by 10-15 bases of correct sequence on both sides, and have a melting
temperature ≥ 78°C.
Mutant Strand Synthesis Reaction:
* Ensure that miniprepped plasmid came from a dam+ strain (DH5α is good).
Prepare the following 50 µL reaction on ice:
10 µL of 5X Phusion HF or GC Buffer (Try HF first).
1 µL 10 mM dNTPs.
2.5 µL sense primer.
2.5 µL antisense primer.
1.5 µL DMSO.
X µL template DNA (Try 10ng first).
X µL ddH20 (Up to 50 µL)
0.5 µL Phusion DNA polymerase (1.0 unit, ADD LAST).
Gently flick the PCR tube to mix the reaction mixture, and perform a quick spin in the Bio-Rad
microcentrifuge. Put the PCR tube back on ice after spinning down, and preheat the thermal cycler
to 98°C before transferring the PCR tube. Cycle the reaction according to the following parameters:
Segment
Cycles
Temperature
Used:
Time
Used:
1
1
98 °C
1 minute
2
18
98 °C
30 seconds
*60-65 °C
50 seconds
72 °C
15-30 sec/kb
3
1
72 °C
10 minutes
4
1
4 °C
Indefinite
* This is experimental, since the polymerase that the QuickChange kit uses tends to need a lower
annealing temperature than our Phusion does. The QuickChange protocol calls for a 60 °C annealing
temperature. We may need to optimize this empirically.
Make sure the PCR reaction has been at 4°C for at least 5 minutes before proceeding. Also,
make sure to have a 37°C incubator ready to go!
Optional: 10 µL of PCR reaction can be checked on a gel. QuickChange protocol says to not worry
about it if bands aren’t visualized at this stage, and to proceed with DpnI digestion in either case.
