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

Generation of SMAD3 Reporter Constructs
The putative enhancer sequence in intron 1 of SMAD3 (Chr15:67,441,598-67,443,643) was first PCR
amplified with primers: forward 5’-GATTGAGCTCCCTGTTCAGCATTTTGAGTTTC-3’ (SacI
restriction site); reverse 5’-GATTGCTAGCGAGCTATTGGAGACTGTGAGA-3’ (NheI restriction site).
PCR was performed on a previously genotyped genomic DNA template that had a CC genotype at
rs17293632 and homozygous for all other SNPs in the amplicon. This PCR product, as well as empty
pGL3-Promoter vector (Promega), were both double-digested with SacI and NheI and subsequently PCR
or gel purified before ligation. Site-directed mutagenesis was used to change the allele at rs17293632
from C to T and performed using the Q5 site-directed mutagenesis kit (New England BioLabs).

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Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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