Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/26: Difference between revisions
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Restriction digest: | Restriction digest: | ||
In two separate tubes, digest the gBlock and the vector using the following reagents. | |||
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Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes. | 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. | |||
Dephosphorylation (Roche) | Dephosphorylation (Roche) | ||
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Finally, transform DH5α using the entire volume of ligated plasmid. Include one negative control (water) and one positive control (original plasmid). | |||
Revision as of 15:28, 26 March 2015
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Entry titleToday I'm going to try restriction digest of the gBlock and the vector, followed by ligation and transformation into DH5α competent E. coli cells. Procedure Restriction digest: 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. Dephosphorylation (Roche)
Final dephosphorylated backbone concentration of 17 ng/μL. Ligation protocol:
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.
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