Single amino acid residue substitutions and site-saturation mutagenesis at sites 149, 205, or 263 were introduced into the native GmDGAT1A. Briefly, the full length of the native GmDGAT1A coding region was cloned into a pGEM R -T Easy Vector (Promega, USA). Site-directed mutagenesis was performed by inverse PCR using PrimeSTAR GXL polymerase (Takara, Japan) and the pGEM R -Tvector as a template. The mutagenic primers were designed to change phenylalanine to leucine (F149L), phenylalanine to valine (F205V), or alanine to valine (A263V). These primer sequences are shown in Supplementary Table S2-2. The full length of the coding region in the pGEM R -T-vector was sequenced and single site mutagenesis was confirmed. To construct double-and triple-site mutants, a single site-mutated GmDGAT1A and a double site-mutated GmDGAT1A were used as a template, respectively. The single site-mutated GmDGAT1A variants were introduced into yeast H1246 and cultured as described above. The HanDGAT1 introduced line was also cultured as an example of highly active DGAT1.
Pgem r t easy vector
Manufactured by Promega
Sourced in United States
The pGEM®-T Easy Vector is a linearized vector with a single 3' terminal thymidine at both ends. This vector is designed for the direct cloning of PCR products generated by thermostable DNA polymerases, which often add a single deoxyadenosine to the 3' ends of the amplified fragments.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pgem r t easy vector
1
Engineered GmDGAT1A Variants via Mutagenesis
2
Site-Directed Mutagenesis of GmDGAT1A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single amino acid residue substitutions and site-saturation mutagenesis at sites 149, 205 or 263 were introduced into the native GmDGAT1A. Briefly, the full length of the native GmDGAT1A coding region was cloned into a pGEM R -T Easy vector (Promega, USA). Site-directed mutagenesis was performed by inverse PCR using PrimeSTAR GXL polymerase (Takara, Japan) and the pGEM R -T-vector as a template. The mutagenic primers were designed to change phenylalanine to leucine (F149L), phenylalanine to valine (F205V), or alanine to valine (A263V). These primer sequences are shown in Supplementary Table S2-2. The full length of the coding region in the pGEM R -Tvector was sequenced and single site mutagenesis was confirmed. The single site mutated GmDGAT1A variants were introduced into yeast H1246, and cultured as described above. The HanDGAT1 introduced line was also cultured as an example of highly active DGAT1.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!