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

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'''SURVEYOR assay'''
'''SURVEYOR assay'''<br>
Assistance from Week 2 Team 1


PCR primers [Rene, please add this info]
PCR primers [Rene, please add this info]
* Forward = ???, Reverse = ???
* Forward = q1F, Reverse = P3R
* Amplicon length = ???
* Amplicon length = 329 bp
* Annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit


PCR templates
 
'''PCR templates'''
# CRISPR "1"
# CRISPR "1"
# CRISPR "2"
# CRISPR "2"
# CRISPR "4"
# CRISPR "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




Revision as of 09:23, 5 August 2014

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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


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




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