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KLD Site-Directed Mutagenesis Protocol

Site-Directed Mutagenesis using WVA, back-to-back primers and KLD
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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***.

Disadvantages

  • 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.

Back-to-back primer site-directed mutagenesis with KLD

STEP 1 - Primer Design

Point Mutation

As in the name of the technique and in contrast to QuickChange and Fast Mutagenesis primer design, both primers must be designed ‘back-to-back’ in opposite directions. Only one (1) of the primers must contain the desired point mutation.

Back-to-back SDM with KLD - point mutation primer design

Deletion

The back-to-back primer design allows for deletions of unlimited size to be generated simply by positioning both 5′ ends of forward and reverse primers directly on the sequence flanking the desired deletion.

Back-to-back SDM with KLD - deletion primer design

Insertion

Both primers must be designed ‘back-to-back’ in opposite directions and either one of them must contain the sequence to be inserted on its 5′ end. The insertion can be split between both forward and reverse primers if desired.

Back-to-back SDM with KLD - insertion primer design

STEP 2 - PCR

WVA PCR Protocol for KLD Mutagenesis

Classic WVA PCR Setup

  • H2O : to 25 ul
  • 5x buffer : 5 ul
  • dNTPs (2.5 mM): 2 ul (0.2 mM final)
  • Forward Primer (10 uM): 0.5 ul (200 nM final)
  • Reverse Primer (10 uM): 0.5 ul (200 nM final)
  • plasmid DNA : 1-10 ng
  • FastPfu FLY (2.5 u/ul) : 0.5 ul

PCR Cycling for WVA

  • Denaturation: 120s at 95 °C
  • 16-25 x
    1. Denaturation: 20s at 95 °C
    2. Annealing: 20s at Tm °C
    3. Extension: 10-30 s/kb at 68 °C
  • Final extension: 600s at 68 °C

Want to go Faster and Save Time?

Use the Fast & Steep PCR protocol for whole vector amplification (WVA) to limit the number of PCR cycles to a minimum and achieve high-yield PCR. The risk of introducing undesired mutations drops drastically by at least 100 000-fold.

STEP 3 - KLD Treatment

KLD : Removal of Methylated Template, Phosphorylation and Ligation

Mix 6 ul sterile ddH2O Save & Exit

+ 1 ul of PCR product

+ 2 ul of 5x KLD buffer

+ 1 ul of KLD mix at RT for 5-15 min.

Incubate at room temperature for 5-15 min.

STEP 4 - Transformation

Transformation of Competent Cells

  1. Thaw 50 ul of high-efficiency (> 108 cfu/ug) chemically competent cells on ice.
  2. Add 1-2 ul of the KLD reaction from Step 3 and gently flick the tube 3 times before incubating on ice for 30 min.
  3. Heat shock the cells by 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 50-200 ul on a prewarmed LB-agar plate containing the appropriate antibiotic(s).

Optimal - Transformation in DMT Competent Cells

Add 1-5 μl of the KLD reaction to 50 μl of DMT chemically competent cells (CD511).
• Incubate on ice for 30 minutes.
• Heat shock at 42°C for 45 seconds.
• Incubate on ice for 2 minutes.
• Add 450 μl SOC or LB, gently shake at 37°C for 1 hour.

Spread 20–200 μl onto appropriate selection plate, incubate overnight at 37°C

KLD Site-Directed Mutagenesis Protocol using Back-to-Back Primers

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