Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/26
|Cloning DBN001_pSB1A3 (1st attempt)||Main project page|
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Today I'm going to try restriction digest of the gBlock and the vector, followed by ligation and transformation into DH5α competent E. coli cells.
In two separate tubes, digest the gBlock and the vector using the following reagents.
Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes.
Dephosphorylate the backbone using an alkaline phosphatase.
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.