Fast Mutagenesis Protocol with FM111Site-Directed Mutagenesis using WVA and partially overlapping primers Get the Fast Mutagenesis KitGo to the Main SDM page
TransBionovo's Fast Mutagenesis Protocol
- 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***.
- 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.
STEP 1 - Primer Design
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
- Denaturation: 20s at 95 °C
- Annealing: 20s at 55 °C
- 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)
- Thaw 50 ul of high-efficiency (> 108 cfu/ug) DMT chemically competent cells on ice.
- 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.
- Heat shock the cells by incubating at precisely 42 °C for 45 s.
- Incubate on ice for > 2 min.
- Add 250 ul of SOC or LB media to the cells, then agitate at 225 rpm, 37 °C for 60 min.
- Spread 200 ul on a prewarmed LB-agar plate containing the appropriate antibiotic(s).