New England Biolabs
KLD enzyme mix
M0554S
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Site-directed mutagenesis is one of the essential techniques in the laboratory. KLD mix offers a combination of three enzymes (Kinase-Ligase-DpnI) in one vial. Together, these three enzymes allow for the rapid circularization of the PCR product and removal of the template DNA, resulting in high-efficiency transformation. Here I describe the method of using this enzyme mix.
Site directed mutagenesis
PCR product
Do not add more than 5 µl of the KLD reaction (PCR product + KLD mix) to 50 µl of competent cells.
Mutations were introduced at the specific sites by using designed primer pair. 1 µl of the PCR products were mixed with 5 µl of KLD reaction buffer, 3 µl of water and 1 µl of KLD mix. The reaction was mixed well and incubated at room temperature (25°C) for 5 minutes. 5 µl of the KLD reaction mixture was transferred to 50 µl of E. coli competent cells and transformation was carried out. Colonies were screened the next day. Three out of four colonies had the correct mutations.
N/A
High efficiency. Easy and time saving.
Expensive
A very efficient enzyme mix that can save time and can greatly improve the site-directed mutagenesis results.