Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/18
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The goal of this project is to change the reporter gene in a stably transfected cancer cell line. The current reporter gene is luciferase, which has poor resolution on a cellular level and requires a time-intensive assay to measure. We intend to replace the luciferase gene and promoter with cyan fluorescent protein (CFP) for rapid measurement of reporter gene activity. We'll also be replacing the luciferase TK promoter with a HPK promoter, which should enhance transcription.
Here's the overall direction this project is going to take:
DBN002 & DBN003 PCR
Primers and gBlock have already been ordered, and primers arrived yesterday. Today I made 100μM stocks of the primers and used the primers to amplify the HPK sequence (with Kozak) and the CFP sequence.
PCR kit used was GoTaq clear 2x Master Mix, containing polymerase, buffer, and dNTPs.
Four reactions were performed. Rxn 1 used KAH45 plasmid as the template (from BioBricks box) and DBN002 f1 and DBN002 r1 as the primers. Rxn 2 used KAH104 plasmid as the template (from BioBricks box) and DBN003 f1 and DBN003 r1 as the primers. Each reaction also had a control reaction containing primers but no template.
PCR program used is named DBN031815. 30 cycles were performed.
The DBN002 primers anneal at 59°C and the DBN003 primers anneal at 68°C.