KLD Site-Directed Mutagenesis Protocol using Back-to-Back Primers
Advantages of KLD Site-Directed Mutagenesis
- Simple to use KLD mix
- High-yield exponential PCR.
- Simple primer design.
- In vitro digestion of the template DNA.
- 90% of clones contain the desired mutation.
- Suitable for insertions of ∼ 80 bases or longer if using oligos > 60-mers.
- Ideal for creating large deletions.
- Easy 3-step workflow***.
- Long PCR protocol – 25 cycles (between 4 and 8 hours or 1 to 2 hours using Fast & Steep PCR).
- Only 1 primer contains the mutation which may generate non-methylated and non-mutated PCR products.
- Primers or Dpn I-generated fragments are likely to be inserted at the ligation site.
***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies, such as the herein KLD strategy, which 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.