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

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(Autocreate 2015/11/27 Entry for Haynes_Lab:Notebook/HPK-CFP_insertion_into_Gal4EED/Luc_using_CRISPR)
 
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==Overview==
==Overview==


Insert content here...
New oligo bridges arrived. Trying a ligase cycling reaction that will include the pJET1.2 plasmid into the product.
 
==Methods==
 
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.
# 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).
 
'''PNK reaction'''
{| {{table}}
|-
| Reagent || Volume
|-
| Clean PCR or dsDNA || 5 parts, 2 µL per part
|-
| 10x T4 Ligation buf (NEB) || 2.0
|-
| T4 PNK (NEB) || 0.5
|-
| dH<sub>2</sub>O || 7.5 μL
|-
| &nbsp; || 20.0 μL
|}
 
* Incubate at 37°C/ 30 min.
* Heat-inactivate PNK at 65°C/ 20 min.




Revision as of 16:19, 27 November 2015

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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. Using a 20 µL reaction volume for the PNK reaction.
  3. 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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