Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/26

Cloning DBN001_pSB1A3 (1st attempt)

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Overview Today 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. Product gBlock pSB1A3-GFP DNA 100ng (5µL) 600ng (3µL) 10x FastDigest Buffer 3 3 EcoRI 1 1 SpeI 1 1 Water 20 22 Total 30 30 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) Reagent Volume DNA (clean digest) up to 17 μL (340 ng) 10x buffer d.p. 2.0 phosphatase 1.0 dH2O ---   20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 17 ng/μL Ligation protocol: Component Volume 10x Roche DNA Ligase Buffer 2 Vector DNA 50ng (3 μL) Insert DNA 37.5ng (12 μL) water to 20uL NEB T4 DNA Ligase 1 Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes. Finally, transform DH5α using the entire volume of ligated plasmid. Include one negative control (water) and one positive control (original plasmid).