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.
Plasmids pEGFP-PI4KA (kindly provided by G. Hammond, NICHD, National Institutes of Health, Bethesda, MD, USA) (, ), p3A-myc (CVB3) (), and p3A-myc (EMCV) () were described previously. Plasmid pM16.1 contains the full-length infectious cDNA sequence of EMCV, strain mengovirus. Plasmid pRLuc-QG-M16.1 was obtained by introducing the luciferase gene upstream of the capsid coding region () in pM16.1. Infectious clones EMCV-3A-A32V and RLuc-EMCV-3A-A32V were generated by introducing the A32V mutation into the 3A sequence of pM16.1 and pRLuc-QG-M16.1, respectively, using mutagenesis primers designed with the NEBaseChanger tool and a Q5 site-directed mutagenesis kit (New England Biolabs) according to the manufacturer’s instructions. EMCV-3A-A34V and RLuc-EMCV-3A-A34V were generated as described for the 3A-A32V mutants. Constructs p3A-A32V-myc and p3A-A34V-myc were generated from p3A-myc (EMCV) using the same strategy. Subgenomic replicons pRib-LUC-CB3/T7 and pRib-LUC-CB3/T7-3A-H57Y, encoding Firefly luciferase in place of the capsid-coding region, were described previously (, ).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Site Directed Mutagenesis (SDM) Hamster - Point mutation BHK-21 PI4KA using Q5® Site-Directed Mutagenesis Kit from New England BioLabs.

Manufacturer protocol

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

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Site Directed Mutagenesis (SDM) Hamster - Point mutation BHK-21 PI4KA using Q5® Site-Directed Mutagenesis Kit from New England BioLabs. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.