Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/14
|Cloning DBN001_pSB1A3, 2nd attempt||Main project page|
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My previous attempt at inserting the gBlock into a vector was unsuccessful. I'm trying again with a different vector, otherwise keeping the protocol the same.
mCherry vector concentration is 74 ng/µL. gBlock concentration is 311 ng/µL.
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 digested vector using an alkaline phosphatase.
Incubate at 37°C for 10 min, then deactivate phosphatase at 75°C for 2 min. Final DNA concentration is 17 ng/μL.
Ligate using Roche DNA Ligase buffer and NEB T4 Ligase.
Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.
Set aside 2µL of ligated plasmid for gel electrophoresis. Transform cells with entire remaining volume of ligated plasmid. Remember to use negative and positive controls (water and untreated vector, respectively).
1kb+ Ladder | digested vector | digested insert | undigested insert | ligation product
Can't really make anything out on the digested gBlock or ligation samples. Too diluted. Might try running another gel with remainder of these fractions.