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QuickChange Site-Directed Mutagenesis Protocol

Site-Directed Mutagenesis using WVA and complementary primers
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QuickChange Site-Directed Mutagenesis

Advantages

  • Both primers contain the mutation.
  • In vitro removal of methylated DNA (parental template).
  • Simple 3-step workflow***.

Disadvantages

  • Long amplification protocol – 25 cycles (between 4 and 8 hours).
  • Low yield amplification of DNA.
  • Complementary primers anneal together.
  • Tandem primer insertion at or near the mutated site often occurs.
  • Large insertions are problematic.
  • Large deletions are problematic.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies that use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

QuickChange Site-Directed Mutagenesis Workflow

STEP 1 - Primer Design

QuickChange Primer Design

Complementary Primers contain the desired mutation at their center

QuickChange Primers

STEP 2 - PCR

QuickChange PCR Protocol

QuickChange PCR Setup

  • H2O : to 50 ul
  • 5x buffer : 10 ul
  • dNTPs (2.5 mM): 4 ul (0.2 mM final)
  • Forward Primer (10 uM): 1.25 ul (250 nM final)
  • Reverse primer (10 uM): 1.25 ul (250 nM final)
  • plasmid DNA : 50 ng
  • FastPfu FLY (2.5 u/ul) : 1 ul

QuickChange PCR Cycling

  • Denaturation: 120s at 95 °C
  • 16-25 x
    1. Denaturation: 20s at 95 °C
    2. Annealing: 30s at 55 °C
    3. Extension: 60 s/kb at 68 °C
  • Final extension: 300s at 68 °C

STEP 3 - Digestion

Removal of Methylated Template

Add 1 ul of DMT Enzyme or DpnI and incubate your at 37 °C for 1 h.

STEP 4 - Transformation

Transformation of Competent Cells

  1. Thaw 50 ul of high-efficiency (> 108 cfu/ug) chemically competent cells on ice.
  2. Add 0.5 – 5 ul of the QuickChange reaction from Step 3 and gently flick the tube 3 times before incubating on ice for 30 min.
  3. Heat shock the cells by incubating at precisely 42 °C for 30-45 s (depends on the cells).
  4. Incubate on ice for > 2 min.
  5. Add 450 ul of SOC or LB media to the cells, then agitate at 200 rpm, 37 °C for 45-60 min.
  6. Spread 100-200 ul on a prewarmed LB-agar plate containing the appropriate antibiotic(s).
QuickChange Site-Directed Mutagenesis Protocol

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