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Fast Mutagenesis Protocol with FM111

Site-Directed Mutagenesis using WVA and partially overlapping primers
Get the Fast Mutagenesis KitGo to the Main SDM page

TransBionovo's Fast Mutagenesis Protocol

Advantages

  • Semi-exponential amplification PCR.
  • Both primers contain the mutation.
  • In vitro digestion and in vivo degradation of non-mutated parental DNA.
  • 95% of clones contain the desired mutation.
  • Suitable for insertions of ∼ 45 bases.
  • Suitable for large deletions.
  • Easy 3-step workflow***.

Disadvantages

  • Long PCR protocol – 25 cycles (between 4 and 8 hours).
  • Partially complementary primers may anneal together.
  • Tandem primer insertion at or near the mutated site may occur.
  • Large deletions may be challenging because of the partial overlap between primers.

***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.

1rst Fast & Steep PCR - lab book

STEP 1 - Primer Design

Fast Mutagenesis FM101 Primer Design

Partially overlapping/complementary

  • One primer must contain the desired mutation at its center.
  • The primer from the opposite strand contains the mutation at the extreme 3′ end.

Fast Mutagenesis Primer

 

STEP 2 - PCR

Fast Mutagenesis System PCR Protocol

Fast Mutagenesis PCR Setup

  • H2O : to 50 ul
  • 2x FastPfu or FastPfu FLY PCR Supermix : 25 ul
  • Forward Primer (10 uM): 1 ul (200 nM final)
  • Reverse Primer (10 uM): 1 ul (200 nM final)
  • plasmid DNA : 1-10 ng

Fast Mutagenesis PCR Cycling

  • Denaturation: 2-5 min at 95 °C
  • 20-25 x
    1. Denaturation: 20s at 95 °C
    2. Annealing: 20s at 55 °C
    3. Extension: 2-4 kb/min at 72 °C
  • Final extension: 10 min at 72 °C

STEP 3 - Digestion

1rst Digestion of Methylated Template (DMT)

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

STEP 4 - Digestion & Transformation

Transformation of DMT Competent Cells and 2nd Digestion of Methylated Template (DMT)

  1. Thaw 50 ul of high-efficiency (> 108 cfu/ug) DMT chemically competent cells on ice.
  2. Add 2 – 5 ul of the DMT 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 45 s.
  4. Incubate on ice for > 2 min.
  5. Add 250 ul of SOC or LB media to the cells, then agitate at 225 rpm, 37 °C for 60 min.
  6. Spread 200 ul on a prewarmed LB-agar plate containing the appropriate antibiotic(s).
Fast Mutagenesis System Protocol

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