Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/26: Difference between revisions
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# Dephosphorylate the backbone using an alkaline phosphatase. | # Dephosphorylate the backbone using an alkaline phosphatase. | ||
Dephosphorylation (Roche) | |||
{| {{table}} cellspacing="3" <!-- Dephos table --> | |||
|- | |||
| bgcolor=#cfcfcf | Reagent | |||
| bgcolor=#cfcfcf | Volume | |||
|- | |||
| DNA (clean digest) || up to 17 μL (340 ng) | |||
|- | |||
| 10x buffer d.p. || 2.0 | |||
|- | |||
| phosphatase || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || --- | |||
|- | |||
| || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL | |||
|} | |||
Final dephosphorylated backbone concentration of 17 ng/μL. | |||
Revision as of 12:15, 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:
Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes.
Dephosphorylation (Roche)
Final dephosphorylated backbone concentration of 17 ng/μL.
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