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

Recombinant His-ISG20 proteins expression and purification
The ORF of wildtype ISG20 and ISG20 mutant were PCR amplified from plasmid F-ISG20 and F-ISG20, respectively, and cloned into pRSET A prokaryotic expression vector (Thermo Fisher Scientific) under the T7 promoter at BamHI and EcoRI sites to generate plasmid expressing His-tagged ISG20 (His-ISG20) and ISG20 (His-ISG20). ISG20 deletion mutant His-ISG20ΔExoI, His-ISG20ΔExoII, and His-ISG20ΔExoIII were derived from His-ISG20 by using Q5 Site-Directed Mutagenesis Kit (New England Biolabs) per the manufacture’s protocol. The protein expression vectors were transformed into BL21(DE3) pLysS Competent Cells (Promega) and the cells were propagated with aeration at 37°C in 0.5 L of SOB broth in the presence of 100 μg/ml Ampicillin to an A of ∼0.6, followed by adding 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) to induce protein expression at 37°C for 3 hours. The induction of the recombinant proteins were detected SDS-PAGE and Coomassie staining as compared to an uninduced control sample.

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) Human - Point mutation Huh7 ISG20 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) Human - Point mutation Huh7 ISG20 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.