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

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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. 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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