Site-directed mutagenesis protocol (adapted from QuickChange)

A protocol for changing one (or a few) bases on a plasmid

SUPPLIES:

Primer Design:

Use the following website to design the QuickChange primers:
http://www.bioinformatics.org/primerx/cgi-bin/DNA_1.cgi

Equipment:

• Thermocycler
• 37°C Incubator

Consumables:

• PCR tubes
• Reagents in table below
• Dpn1 restriction enzyme (to remove template from rnx)

Buffers and Solutions:

QuickChange rxn:

Reagent	Amount / L
DNA template (~5ng/µl)	2.0 µl
5x HF buffer	10 µl
10 mM dNTPs	1 µl
10µM Forward Primer	2.5 µl
10µM Forward Primer	2.5 µl
100% DMSO	2.5 µl
50 mM MgCl2	1 µl
Phusion Polymerase	0.5 µl
H2O	28 µl
TOTAL	50 µl

PROTOCOL

Thermocycler settings:

1. ) 98C 30 sec
2. ) 98C 30 sec
3. ) 55C 1 min (can be empirically optimized)
4. ) 72F 1 min/kb (can change time for your polymerase of choice)
   • repeat steps 2-4 x12-18
5. ) hold at 10C

add 1µl Dpn1 to each tube and mix thoroughly. Incubate for 1hr at 37C (thermocycler or incubator). Proceed to transformation or store at -20C.

-- Main.MattMcGuffie - 25 Oct 2018