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

Plasmids
pT2/HB transposon vector was purchased from Addgene (plasmid #26557). First, the puromycin resistance gene with corresponding PGK promoter elements was amplified from pLKO.1puro vector (purchased from Addgene, plasmid #8453) and cloned into the pT2/HB vector using EcoRI and HindIII restriction sites, yielding pT2/HB-puro. Second, ABCA3 cDNA fused to a C-terminal HA-tag was amplified with corresponding cytomegalovirus (CMV) promoter elements from pcDNA4TO-ABCA3-HA vector and cloned into pT2/HB-puro using NheI and NotI restriction sites, yielding pT2/HB-puro-ABCA3-HA. The R288K point variant was introduced into pT2/HB-puro-ABCA3-HA using the Q5® site-directed mutagenesis kit with the following primers: R288K-for 5’-GAGAA GGAAA AGAGG CTGAA GGAGT AC-3’ and R288K-rev 5’-CTGCA CGACA GCACG GGC-3’. pCMV(CAT)T7-SB100 transposase was ordered from Addgene (plasmid #34879). All resulting vector constructs were verified using Sanger sequencing. Sequence analysis and alignment was done using Clone Manager Suite (Version 6.00).

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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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