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

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Final dephosphorylated backbone concentration of 17 ng/μL.
Final dephosphorylated backbone concentration of 17 ng/μL.


Ligation protocol:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10x T4 DNA Ligase Buffer||2
|-
| Vector DNA||50ng
|-
| Insert DNA||37.5ng
|-
| water||to 20uL
|-
| T4 DNA Ligase||1
|}


Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.




Revision as of 12:32, 26 March 2015

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Entry title

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:

  1. In two separate tubes, digest the gBlock and the vector using the following reagents.
Product gBlock pSB143-GFP
DNA 100ng (5uL) 600ng (3uL)
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.

  1. 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] = 25 ng/μL

Final dephosphorylated backbone concentration of 17 ng/μL.

Ligation protocol:

Component Volume
10x T4 DNA Ligase Buffer 2
Vector DNA 50ng
Insert DNA 37.5ng
water to 20uL
T4 DNA Ligase 1

Incubate at room temp. for 10 minutes, then heat inactivate at 65°C for 10 minutes.


Follow the instructions in the gBlock user guide, under 'Cohesive-end restriction cloning'.



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