Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/03/24: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 1: Line 1:
{|{{table}} width="800"
{|{{table}} width="800"
|-
|-
|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;"> DBN002 Gel Extraction & PCR</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"|<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>}}
|-
|-
| colspan="2"|
| colspan="2"|
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### -->
==Entry title==
==Overview==


I cut out the 1500bp band in lane 8 from the agarose gel run yesterday and purified the DNA using the Sigma GenElute Gel Extraction Kit (protocol can be viewed [http://openwetware.org/images/5/58/Sigma_Gel_Extraction_Kit.pdf here]). After extracting the DNA, I performed a thermal gradient PCR on the fragment using the following quantities of reagents:
I cut out the 1500bp band in lane 8 from the agarose gel run yesterday and purified the DNA using the Sigma GenElute Gel Extraction Kit (protocol can be viewed [http://openwetware.org/images/5/58/Sigma_Gel_Extraction_Kit.pdf here]). After extracting the DNA, I performed a thermal gradient PCR on the fragment using the following quantities of reagents:

Revision as of 11:50, 22 April 2015

DBN002 Gel Extraction & PCR <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Overview

I cut out the 1500bp band in lane 8 from the agarose gel run yesterday and purified the DNA using the Sigma GenElute Gel Extraction Kit (protocol can be viewed here). After extracting the DNA, I performed a thermal gradient PCR on the fragment using the following quantities of reagents:

Reagent Volume (uL) for 9 rxns (uL)
2x GoTaq MM 10 90
MB grade H2O 9.6 86.4
forward primer 0.1 0.9
reverse primer 0.1 0.9
template 0.2 1.8
Total 20 180

Thermal cycler program:

Step Temperature °C Duration
Initial denature 95 120 sec
denature 95 30 sec
anneal 59-->66 30 sec
extension 72 90 sec
final extension 72 300 sec
soak 4 indef.

Thermal gradient is from 59°C to 66°C. Program name is DBN20150324.



Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/HPK-CFP_insertion_into_Gal4EED/Luc_using_CRISPR/2015/03/24&oldid=881861"