Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/30

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Overview

Performing LCR on the PNK-treated parts from last week.

Methods

  • Add components to the PNK DNA reaction as described in the table below
  • Set up the reactions in two PCR tubes, 25 µL per tube
Reagent Volume
PNK DNA 20 μL
Oligo Bridges 15.0 (3 each)
10X Ampligase Buffer 5.0
DMSO 4.0
Betaine (5M) 4.5
Ampligase 1.7
dH2O 0 μL
  50.2 μL

Thermal cycler program:

Step Temp (°C) Time (s)
Initial Denaturation 94 120
50 cycles:
Denaturation 94 10
Annealing 55 30
Ligation 66 60
Hold 4

Transformation

Follow the instructions in the protocol described here. Setting up three plates:

  1. Sample
  2. Negative control - pJET vector only
  3. negative control - no plasmid

Notes

May have to perform a PCR clean-up on the sample next time before transforming cells.

Colony PCR Results

Performed colony PCR on the one colony on the sample plate. No product. Giving up on LCR and switching to Golden Gate assembly.