Team:NUS Singapore-Sci/GLB Logbook

NUS Singapore Science: InterLab

Reporter System
GLB1

1. Cloning of the GLB1 from Genescript Plasmid
GLB1 is amplified from the template (Genscript plasmid, pCDNA3.1+C(-K)-DYK) using Q5 DNA polymerase (NEB) via PCR (Fig.1). The PCR protocol used is shown below, the primers used are GLB-HindIII-F and GLB-KpnI-R with annealing temperatures of 56℃, 57℃ and 58℃, and extension time of 1 min. The two primers used during PCR amplification also introduced additional HindIII and KpnI restriction sites at the front and back of GLB1 gene for downstream cloning. This GLB1 construct is addressed as GLB1 WT in subsequent experiments and this construct will be cloned into pEGFP-C1 for mammalian expression and pSB1C3 for parts submission.


Figure 1. PCR amplification of GLB1. GLB1 containing HindIII and KpnI restriction sites (in red box) are amplified during PCR and will be known as GLB1 WT. The size of GLB1 WTI is ~2218 bp and is successfully amplified in all PCR reactions. Lane 1 and 2 are performed under an annealing temperature of 56℃, lane 3 and 4 of 57℃, and lane 5 and 6 of 58℃. The top DNA band (in blue box) represents the original plasmid construct, pCDNA3.1+C(-K)-DYK, from Genscript with a size of ~7400 bp.