Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/03

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Overview

Performing Ligase Cycling Reaction (LCR) today. See instructions here.

Methods

Prep Bridge Oligos

Make 300 nM working solutions of the dsDNA oligo bridges:

  • 3 µL forward strand (100 µM)
  • 3 µL reverse strand (100 µM)
  • 94 µL H2O

Prep DNA fragments

Dilute purified DNA from Phusion PCR to 30 nM:

  • Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
DNA fragment Length (bp) measured ng/µL purified DNA volume (µL) final volume (µL)
HA11001490.6550
HA21001270.7750
EM7-ZeoR4422351.8350
HPK-CFP19722480.1150

Since HPK-CFP fragment is at too low a concentration, will use it straight instead.


PNK Reaction

Use polynucleotide kinase (PNK) to add 5' phosphate groups to DNA fragments.

  • Use 2.0 μL of each diluted dsDNA per 10 μL PNK reaction
    • Use 2.5 µL of HPK-CFP dsDNA
  • NOTE: All DNA to be treated with PNK goes in the same tube!
Reagent Volume
Clean PCR or dsDNA 8.5 μL
10x T4 Ligation buf (NEB) 1.0
T4 PNK (NEB) 0.5
dH2O 0 μL
  10.0 μL
  • Incubate at 37°C/ 30 min.
  • Heat-inactivate PNK at 65°C/ 20 min.

Ligase Cycling Reaction

In a PCR tube, set up the following reaction. Use 3 µL of each dsDNA oligo bridge (9 µL total).

Reagent Volume
PNK DNA 10 μL
dsDNA Oligo Bridge 9.0
10X Ampligase Buffer 3.0
DMSO 2.4
Betaine (5 M) 2.7
Ampligase 1.0
dH2O 1.9 μL
  30.0 μL

Thermal cycler program:

  1. 2 min at 94 °C
  2. 10 s at 94 °C
  3. 30 s at 55 °C
  4. 60 s at 66 °C
  5. Repeat steps 2-5 x50 times
  6. hold at 4 °C



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