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

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Cloning DBN001_pSB1A3 (1st attempt) Main project page
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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
DNA100ng (5µL)600ng (3µL)
10x FastDigest Buffer33
EcoRI11
SpeI11
Water2022
Total3030

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 Buffer2
Vector DNA50ng (3 μL)
Insert DNA37.5ng (12 μL)
waterto 20uL
NEB T4 DNA Ligase1

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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