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
  • 37C 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/l) 2.0 l
5x HF buffer 10 l
10 mM dNTPs 1 l
10M Forward Primer 2.5 l
10M Forward Primer 2.5 l
100% DMSO 2.5 l
50 mM MgCl2 1 l
Phusion Polymerase 0.5 l
H2O 28 l
TOTAL 50 l

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 1l 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

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Topic revision: r1 - 25 Oct 2018 - 20:12:41 - Main.MattMcGuffie
 
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