Haynes Lab:Notebook/CRISPR Editing/2014/08/05: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 41: Line 41:
'''PCR product clean-up'''
'''PCR product clean-up'''
* Used QIAquick PCR Purification kit
* Used QIAquick PCR Purification kit
* Elute with 50 μL H<sub>2</sub>O


{| {{table}}
{| {{table}}
Line 50: Line 51:
| CRISPR "2" || ### || ### || ###
| CRISPR "2" || ### || ### || ###
|-
|-
| CRISPR "3" || ### || ### || ###
| CRISPR "4" || ### || ### || ###
|-
|-
| CRISPR "4" || ### || ### || ###
| Blank 1 || ### || ### || ###
|-
| Blank 2 || ### || ### || ###
|-
| Blank 3 || ### || ### || ###
|}
|}
* Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume




Line 60: Line 67:
** IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation
** IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation
** Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
** Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
* 200 ng in final volume of 10 μL
* 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 15 μL


# 100 ng CRISPR "1" + 100 ng Blank (IDT approach)
# 100 ng CRISPR "1" + 100 ng Blank (IDT approach)

Revision as of 11:06, 5 August 2014

Project name <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

08/05/14

  • SURVEYOR assay - for mCherry CRISPR
  • qPCR melt curve optimization - mCherry CRISPR 1


SURVEYOR assay
Assistance from Week 2 Team 1

PCR primers [Rene, please add this info]


PCR templates

  1. CRISPR "1"
  2. CRISPR "2"
  3. CRISPR "4"
  4. "Blank" (3x)


PCR reaction

  • Reactions: 20 ng template DNA, 1x HF Phusion buffer, 0.2 mM dNTPs, 2.5 μL of 10 μM for each primer, 0.5 μL Phusion polymerase; in 50 μL final volume
  • Program
    • 98°C/ 30 sec
  • 30x [98°C/ 10 sec, 58°C/ 15 sec, 72°C/ 15 sec]
  • 72°C/ 10 min
  • 4°C hold


PCR product clean-up

  • Used QIAquick PCR Purification kit
  • Elute with 50 μL H2O
Sample OD 260 260/280 ng/uL
CRISPR "1" ### ### ###
CRISPR "2" ### ### ###
CRISPR "4" ### ### ###
Blank 1 ### ### ###
Blank 2 ### ### ###
Blank 3 ### ### ###
  • Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume


Mismatch annealing

  • Testing 2 methods
    • IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation
    • Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway
  • 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 15 μL
  1. 100 ng CRISPR "1" + 100 ng Blank (IDT approach)
  2. 200 ng CRISPR "1" (Zhang approach)
  3. 200 ng Blank (wild type control)


qPCR melt curve optimization - mCherry CRISPR 1
Assistance from Kumaran Lleng

Optimization parameters

  • Borun et al reported cleaner peak profiles after a 30-cycle amplification (vs. 40 cycles)
    • We will use 30 cycles instead of 45
  • Borun et al used at LEAST 50 ng genomic DNA in their reactions (final amount per PCR ambiguous)
    • We will test 20 ng, 50 ng, and 70 ng (70 is our upper limit because of the concentration of the Blank DNA sample)


Master reaction list

Rxn # Template Target (primers) Method
1 1 20 ng q2F_q3R CNV melt
2 1 20 ng GAPDH B2 "
3 1 50 ng q2F_q3R "
4 1 50 ng GAPDH B2 "
5 1 70 ng P3F_q3R "
6 1 70 ng GAPDH B2 "
7 Blank 20 ng q2F_q3R "
8 Blank 20 ng GAPDH B2 "
9 Blank 50 ng q2F_q3R "
10 Blank 50 ng GAPDH B2 "
11 Blank 70 ng q2F_q3R "
12 Blank 70 ng GAPDH B2 "

Notes:

  • Opaque white plates
  • Final reaction volumes = 15 μL
  • Each reaction had 3.0 μL of 750 nM F/R primers, 20 ng - 70 ng template DNA, 1x Roche SYBR
  • Batch (master) mixes...
    • Primers: Primers plus SYBR, 10.5 μL mix per relevant well, one mix per unique primer pair
    • Templates: DNA plus water, 4.5 μL mix per relevant well, one mix per unique template
    • Final triplicates for the 96-well plate were first made as a single batch, then aliquoted from the batch tube into the 96-well plate
  • nTc for q2F_q3R and GAPDH confirmed no background signal under identical conditions on 08/04/14


Run PCR - Bio-Rad CFX96

  • 95°C, 3 min
  • 30x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)]
  • 72°C, 30 sec
  • Melt: 60°C -- +0.1°C/ 5 sec (measure) --> 95°C




Retrieved from "https://openwetware.org/mediawiki/index.php?title=Haynes_Lab:Notebook/CRISPR_Editing/2014/08/05&oldid=805149"