Site Directed Mutagenesis Troubleshooting & FAQs

Site Directed Mutagenesis Kit Troubleshooting & FAQs

Site-directed mutagenesis (SDM), sometimes referred to as site-specific or directed mutagenesis, is a method of altering the nucleotide sequence of a gene at a specified location. SDM can be used to introduce specific nucleotide substitutions (or deletions) in a controlled manner. This online site directed mutagenesis guide provides researchers with comprehensive solutions and suggestions to support SDM challenges.

Problem Possible Solution

Plasmids cannot be amplified.

Primer design is wrong: re-check the primer design.

The amplification reaction mixture is not correctly prepared: repeat experiment.

The amplification reaction is not optimized: the concentration of Mg2+, the amount of enzyme and the amplification program can be optimized.

The quality of template is not good: long-term storage, repeated freezing and thawing can cause the breakage, open-loop or degradation of the plasmids. Please use freshly prepared plasmids as templates.

There are no or few colonies on the plate.

The efficiency of the competent cell is very low. Use freshly prepared competent cells or competent cells stored properly and make sure the transformation efficiency of competent cells is more than 10⁷ cfu/μg.

The amount of DNA or ratio of fragments is suboptimal in the recombination reaction. Add the amount of DNA as recommended. Check the concentration of DpnI treated product. DNA concentration must be measured agarose gel electrophoresis and not by any other method such as an absorbance assay.

The DNA in the recombination cyclization contains impurities inhibiting the reaction; or the volume of unpurified DpnI treated product is more than one fifth of total volume. Perform gel extraction of DpnI treated products. Try to avoid complexing agent (e.g. EDTA) in the recombination reaction. Therefore, we recommend that the purified DNA should be dissolved in ddH2O of pH 8.0 instead of TE buffer.

Addition of too much DNA to the competent cells: the volume of DNA should not exceed 1/10 the volume of competent cells, otherwise it will reduce the transformation efficiency.

The transformation inhibitory effect occurs: High concentration of input DNA can inhibit the transformation. In this case, one fifth of the DNA should be used for transformation.

Incorrect site-directed mutation

The primers are not designed correctly. Check the primer design.

The template plasmids are not methylated. DpnI can only recognize the methylated DNA. Purify the template plasmids from the host strains with functional methylases.

Too much plasmid used as template. For most plasmids, 1 ng of DNA is enough template for the PCR reaction. Too much plasmid will lead to incomplete digestion by DpnI, which reduces the successful rate of mutation introduction.

Mutations at non-target site.

The template plasmid carries some unknown mutations: confirm the sequence of the template.

Too many number of amplification cycles: to prevent non-target mutations during the amplification, the number of amplification cycle should not exceed 30 when the amplification efficiency is good.

Other Notes:

a) When choosing the reverse complementary region of primers, avoid repetitive sequences. When GC content is 40% to 60%, cyclization recombination efficiency is maximized. If the GC content is higher than 70% or less than 30%, the cyclization efficiency will be significantly inhibited.
b) The double-base mutation strategy can also be used for single base mutations (one of the two sites will not undergo base modification). Therefore, if the amplification cannot be carried out in single base mutation, try to use the double base mutation strategy