Q5® Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Protocol tips
- Only use 1 μl of PCR product in the KLD reaction. Carrying too much PCR product forward can decrease transformation efficiency. - Only use 5 μl of the KLD reaction in the transformation. If more KLD reaction is added, a buffer exchange step, such as PCR purification, should be included prior to transformation.

Publication protocol

For generating SBP constructs, SBP was amplified from the pTrAP vector [34] and subcloned via BamHI and NotI sites into the pEGFP-N2 vector, replacing GFP with SBP. To generate the ΔNTD mutant of human TMEM120A-SBP (lacking amino acids 2–105), site directed mutagenesis was done using the NEB Q5 mutagenesis kit strategy with minor modifications. Briefly, non-phosphorylated primers were used (forward 5’-GGATTGTACCTGAGCCTGGTTCTG and reverse 5’-CATGGGTCGAGATCTGAGTCCG) in a PCR reaction with Phusion polymerase and TMEM120A-SBP plasmid. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells. All the constructs used in this study were sequenced at the regions of interest using Sanger DNA sequencing.



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Manufacturer protocol

Download the product protocol from New England BioLabs for Q5® Site-Directed Mutagenesis Kit below.

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