Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/11/09

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Overview

Repeating LCR, this time using the HPK-CFP fraction before gel purification. Using the steps outlined here and here.

Methods

Prep Bridge Oligos

Done previously


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)
HPK-CFP 1972 236 8.15 50

All other DNA fragments prepped last week.


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
  • NOTE: All DNA to be treated with PNK goes in the same tube!
  • Perform 4 reactions (40 µL total) so we can try other LCRs later if needed
Reagent Volume (µL) for 4 rxns (µL)
Clean PCR or dsDNA 8.0 32.0
10x T4 Ligation buf (NEB) 1.0 4.0
T4 PNK (NEB) 0.5 2.0
dH2O 0.5 2.0
  10.0 40.0
  • 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

PCR on LCR fragment

Reaction volume: 75 µL (split into 3 tubes to be pooled later)

Going to try using Taq polymerase because of its 5'-->3' exonuclease activity to cleave oligo bridges. Hypothesize that Phusion's lack of strand displacement or exonuclease activity is what caused previous PCR to fail.

Reagent Stock Volume used (µL)
GoTaq 2x MM 2x 37.5
Primer (for) 100 µM 0.75
Primer (rev) 100 µM 0.75
Template 50 ng/µL 0.75
H2O - 35.25
Total 75

Reactions:

Reaction Forward primer Reverse primer template
DBN008 P40 P41 LCR product

All primers have annealing temperature at 60°C

Thermal cycler program:

STEP TEMP TIME
Initial Denaturation 95°C 30 seconds
30 Cycles 95°C 15 seconds
60°C 30 seconds
68°C 150 seconds
Final Extension 68°C 5 minutes
Hold 4°C infinite

Results

Gel image:

File:2015-11-10 pMaxGFP LCR unamplified PCR of LCR.tif

From left to right: 1kb+ ladder, pMaxGFP, LCR (unamplified), PCR of LCR (3 lanes)

Looks like I have product! Expected size is ~2500bp, and there's bands a little bit above the 2000bp band in the ladder. Weak though. Primers seem to have a stronger affinity for something else... primer dimerization going on perhaps? Or the dye in the GoTaq green master mix is fluorescing right there. That seems likely, actually. Will have to try again, using GoTaq clear master mix, and give myself time to do gel extraction.