Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/14

Cloning DBN001_pSB1A3, 2nd attempt

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Overview 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. Major steps: ✓ Restriction digest the vector and insert with EcoRI and SpeI followed by heat inactivation. ✓ Dephosphorylate the backbone followed by heat inactivation. ✓ Ligate using NEB T4 DNA ligase. ✓ 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 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. Product DBN001 mCh_pSB1A3 DNA 100ng (0.3 µL) 600ng (8.1µL) 10x FastDigest Buffer 3 3 EcoRI 1 1 SpeI 1 1 Water 24.7 16.9 Total 30 30 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) Reagent Volume DNA (clean digest) up to 17 μL (340 ng) 10x buffer d.p. 2.0 phosphatase 1.0 dH2O --- Total 20 µL 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. Component Volume 10x Roche DNA Ligase Buffer 2 Vector DNA 50ng (3 μL) Insert DNA 37.5ng (11.25 μL) water 2.75 µL NEB T4 DNA Ligase 1 Total 20 µL 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.