Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/27: Difference between revisions

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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</span>
|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Today's project is...</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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Mostly taken from [http://openwetware.org/wiki/Haynes:LCR_Assembly here]. Only major differences:
Mostly taken from [http://openwetware.org/wiki/Haynes:LCR_Assembly here]. Only major differences:
# Prepared diluted oligo bridge stocks, as the previous stocks I was using were 10x more concentrated than called for in the protocol.
# Prepared diluted oligo bridge stocks, as the previous stocks I was using were 10x more concentrated than called for in the protocol.
# pJET1.2 plasmid is at 50 ng/µL, which is slightly below 30 nM concentration. Will be using straight plasmid for this reaction.
# Using a 20 µL reaction volume for the PNK reaction.
# Using a 20 µL reaction volume for the PNK reaction.
# Using a 45 µL reaction volume for the LCR reaction (will do this on Monday).
# Using a 45 µL reaction volume for the LCR reaction (will do this on Monday).

Latest revision as of 01:22, 27 September 2017

Today's project is... Main project page
Previous entry      Next entry

Overview

New oligo bridges arrived. Trying a ligase cycling reaction that will include the pJET1.2 plasmid into the product.

Methods

Mostly taken from here. Only major differences:

  1. Prepared diluted oligo bridge stocks, as the previous stocks I was using were 10x more concentrated than called for in the protocol.
  2. pJET1.2 plasmid is at 50 ng/µL, which is slightly below 30 nM concentration. Will be using straight plasmid for this reaction.
  3. Using a 20 µL reaction volume for the PNK reaction.
  4. Using a 45 µL reaction volume for the LCR reaction (will do this on Monday).

PNK reaction

Reagent Volume
Clean PCR or dsDNA 5 parts, 2 µL per part
10x T4 Ligation buf (NEB) 2.0
T4 PNK (NEB) 0.5
dH2O 7.5 μL
  20.0 μL
  • Incubate at 37°C/ 30 min.
  • Heat-inactivate PNK at 65°C/ 20 min.



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