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

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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).



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