The TransGen Fast MultiSite-Directed Mutagenesis System is a simple to use and highly efficient kit for generating multiple mutations.
This kit takes advantage of proprietary assembly mix and homologous recombination to achieve directional cloning of PCR fragments that share 15-25 bp overlapping sequences into any linearized vector.
– Fast: 15 minutes.
– Broad: no restriction enzyme digestions. Can be cloned into any sites.
– Kanamycin and Ampicillin resistance genes for choice of selection.
– High efficient: up to 95% cloning efficiency.
– Seamless: no extra sequences introduced; up to 5 frgments assembly.
|Component||FM201-01 (10 rxns)|
|2×TransStart ® FastPfu PCR SuperMix||1 ml|
|DMT Enzyme (10 units/μl)||30μl|
|2×Assembly Mix||50 μl|
|DMT Competent Cell||10×50 μl|
|ddH 2 O||1 ml|
Preparation of multisite mutagenic fragment
(1) Primer Design
• Both primers contain overlapping region at the 5’ ends and extended region at the 3’ ends, with mutation site on overlapping region, as shown in Figure 1.
• Primer length: Both primers (forward and reverse) should be approximately at 25-40 nucleotides in length, excluding the mutation site. Primers should have an overlapping region of 15-20 nucleotides and have an extension region of at least 10 nucleotides.
(2) Preparation of mutated fragment
• The mutation sites are located on one pair of primers, as shown in Figure 2.
• The mutation sites are located on multiple pairs of primers, with F1/R1, F2/R2,….Fn/Rn for amplification, as shown in figure 3.
|Plasmid||1-10 ng||as required|
|Forward Primer (10 μM)||1 μl||0.2 μM|
|Reverse Primer (10 μM)||1 μl||0.2 μM|
|2×TransStart ® FastPfu PCR SuperMix||25 μl||1×|
|ddH 2 O||to 50 μl||Not applicable|
*1. Annealing temperature depends on primers.
*2. We suggest to perform 25 cycles for PCR. For low yield PCR products, we suggest to use up to 30 cycles.
Amplified PCR products may be checked by electrophoresis with 10 μl of PCR product on a 1% agarose gel.
(3) Digestion of PCR Product with DMT
Add 1 μl of DMT enzyme into PCR product, mix thoroughly and incubate at 37 o C for 1 hour.
(4) Purification of PCR products
For PCR product with single expected band, it is suggested to using PCR Purification Kit to purify PCR products; for PCR product with multibands, it is suggested to use Quick Gel Extraction Kit to purify PCR products.
Assembly of Mutated Fragments
|2×Assembly Mix||5 μl|
|Amplified fragment A||x μl*|
|Amplified fragment B||y μl*|
|Amplified fragment N||z μl*|
|ddH 2 O||to 10 μl|
*Suggested amount is 20-150 ng
Gently mix and perform reaction at 50 o C for 15 minutes. After reaction, transfer the reaction onto ice for several seconds.
(1) Add 2 μl of assembly products into 50 μl of DMT Chemically Competent Cell (DNA should be added immediately after thawing the cells on ice) and mix by tapping gently. Incubate on ice for 20-30 minutes.
(2) Heat-shock at 42 oC for exactly 45 seconds, quickly remove from 42 oC water bath and place on ice for 2 minutes.
(3) Add 250 μl of SOC or LB medium (prewarm to room temperature), and incubate at 37 oC for 1 hour with shaking at 200 rpm.
(4) Pre-warm plate (contain proper resistance) at 37 oC in incubator.
(5) Spread 100-200 μl of transformants on the plate and incubate at 37 oC overnight.
Positive Clone Analysis
Analyze the clones by sequencing with proper primer.