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

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Cloning DBN001_pSB1A3 (1st attempt)</span>
|style="background-color: #F2F2F2" align="center"|<html><img src="/images/9/94/Report.png" border="0" /></html> [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>}}
|style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;}}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}}
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==Entry title==
==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.
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>
==Procedure==


Restriction digest:
Restriction digest:
# In two separate tubes, digest the gBlock and the vector using the following reagents.
 
In two separate tubes, digest the gBlock and the vector using the following reagents.
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Product'''
| align="center" style="background:#f0f0f0;"|'''Product'''
| align="center" style="background:#f0f0f0;"|'''gBlock'''
| align="center" style="background:#f0f0f0;"|'''gBlock'''
| align="center" style="background:#f0f0f0;"|'''pSB143-GFP'''
| align="center" style="background:#f0f0f0;"|'''pSB1A3-GFP'''
|-
|-
| DNA||100ng (5uL)||600ng (3uL)
| DNA||100ng (5µL)||600ng (3µL)
|-
|-
| 10x FastDigest Buffer||3||3
| 10x FastDigest Buffer||3||3
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Mix and incubate at 37°C for 15 minutes. Then heat-inactivate by incubating at 80°C for 20 minutes.
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.
Dephosphorylate the backbone using an alkaline phosphatase.


Dephosphorylation (Roche)
Dephosphorylation (Roche)
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| dH<sub>2</sub>O || ---
| dH<sub>2</sub>O || ---
|-
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 17 ng/μL
|}
|}


Final dephosphorylated backbone concentration of 17 ng/μL.


Ligation protocol:
Ligation protocol:
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| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
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| 10x T4 DNA Ligase Buffer||2
| 10x Roche DNA Ligase Buffer||2
|-
|-
| Vector DNA||50ng
| Vector DNA||50ng (3 μL)
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| Insert DNA||37.5ng
| Insert DNA||37.5ng (12 μL)
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| water||to 20uL
| water||to 20uL
|-
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| T4 DNA Ligase||1
| NEB T4 DNA Ligase||1
|}
|}


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Follow the instructions in the [http://openwetware.org/wiki/Image:Gblocks-user-guide.pdf gBlock user guide], under 'Cohesive-end restriction cloning'.
Finally, transform DH5α using the entire volume of ligated plasmid. Include one negative control (water) and one positive control (original plasmid).




Latest revision as of 00:52, 27 September 2017

Cloning DBN001_pSB1A3 (1st attempt) Main project page
Previous entry      Next entry

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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