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

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==Entry title==
==Entry title==
* Insert content here...
 
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.
 
<b>Procedure</b>
 
Restriction digest:
 
In two separate tubes, digest the gBlock and the vector using the following reagents.
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Product'''
| align="center" style="background:#f0f0f0;"|'''gBlock'''
| align="center" style="background:#f0f0f0;"|'''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.
 
Dephosphorylate the backbone using an alkaline phosphatase.
 
Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA (clean digest) || up to 17 μL (340 ng)
|-
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 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 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).




Revision as of 15:28, 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:

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.

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 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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