Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/14: Difference between revisions
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# ✓ Ligate using NEB T4 DNA ligase. | # ✓ Ligate using NEB T4 DNA ligase. | ||
# ✓ Transform DH5α-Turbo with ligated plasmid. Use negative control (water) and positive control (untreated vector). | # ✓ Transform DH5α-Turbo with ligated plasmid. Use negative control (water) and positive control (untreated vector). | ||
# Run an agarose gel containing lanes with: Digested vector, digested insert, ligation product | # ✓ Run an agarose gel containing lanes with: Digested vector, digested insert, ligation product | ||
mCherry vector concentration is 74 ng/µL. gBlock concentration is 311 ng/µL. | mCherry vector concentration is 74 ng/µL. gBlock concentration is 311 ng/µL. | ||
Revision as of 18:12, 14 April 2015
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Ligation of mCherry sender vector + gBlock insertMy 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. Major steps:
mCherry vector concentration is 74 ng/µL. gBlock concentration is 311 ng/µL. --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. --Dephosphorylation-- Dephosphorylate the digested vector using an alkaline phosphatase. Dephosphorylation (Roche)
Incubate at 37°C for 10 min, then deactivate phosphatase at 75°C for 2 min. Final DNA concentration is 17 ng/μL. --Ligation-- 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. --Transformation-- 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). --Gel Electrophoresis-- Lanes: 1kb+ Ladder | digested vector | digested insert | undigested insert | ligation product Gel image:
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. | ||||||||||||||||||||||||||||||||||||||||||||||||
