Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/26: Difference between revisions
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| dH<sub>2</sub>O || --- | | dH<sub>2</sub>O || --- | ||
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| || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = | | || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 17 ng/μL | ||
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Ligation protocol: | Ligation protocol: |
Revision as of 16:59, 26 March 2015
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | |||||||||||||||||||||||||||||||||||||||||||||
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)
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.
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