Haynes Lab:Notebook/CRISPR Editing/2014/08/04: Difference between revisions

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CRISPR 4 - US-specific
CRISPR 4 - US-specific
* Primer pair 1 (q2R_q2F)  
* Primer pair 1 (q2R_q2F)  
** Untreated control Cq_q2R_q2F / Peak_GAPDH =  
** Untreated control Cq_q2R_q2F / Cq_GAPDH =  
** CRISPR treated Peak_q2R_q2F / Peak_GAPDH =  
** CRISPR treated Cq_q2R_q2F / Cq_GAPDH =  
** Conclusion:  
** Conclusion:  
** Notes: no signal from nTc, so good primer pair with no primer dimer signal
** Notes: no signal from nTc, so good primer pair with no primer dimer signal
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CRISPR 4 - US-specific
CRISPR 4 - US-specific
* Primer pair 2 (P3F_q3R)
* Primer pair 2 (P3F_q3R)
** Untreated control Peak_P3F_q3R / Peak_GAPDH =  
** Untreated control Cq_P3F_q3R / Cq_GAPDH =  
** CRISPR treated Peak_P3F_q3R| / Peak_GAPDH =  
** CRISPR treated Cq_P3F_q3R| / Cq_GAPDH =  
** Conclusion:  
** Conclusion:  
** Notes: ''some signal from nTc, not-so-good primer pair''
** Notes: ''some signal from nTc, not-so-good primer pair''
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{|
{|
|-
|-
| Sample || Method || Norm Expt. value || Norm control || Conclusion
| Experiment || Method || Norm Expt. value || Norm control || Conclusion
|-
|-
| "1" - gRNA1/Cas9 || CNV melt || ### || ### || ###
| "1" - gRNA1/Cas9 primer pair 1 || CNV melt || ### || ### || ###
|-
| "1" - gRNA1/Cas9 primer pair 2 || CNV melt || ### || ### || ###
|}
|}


Revision as of 06:55, 5 August 2014

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08/04/14

  • mCherry CRISPR qPCR
  • Gal4EED/luc CRISPR - change growth medium to pen-strep-free, with 1 μg/mL dox for dox+ cells


mCherry CRISPR qPCR
Genomic DNA from CRISPR transfected-cells:

  1. "1" - gRNA1/Cas9
  2. "2" - gRNA2/Cas9
  3. "4" - gRNA4T/Cas9n, gRNA4B/Cas9n
  4. "Blank" - mock transfection with H2O instead of plasmid

Master reaction list

Rxn # Template Target (primers) Method
1 1 q2F_q3R CNV melt
2 1 P3F_q3R CNV melt
3 1 GAPDH B2 CNV melt
4 Blank q2F_q3R CNV melt
5 Blank P3F_q3R CNV melt
6 Blank GAPDH B2 CNV melt
7 nTc q2F_q3R CNV melt
8 nTc P3F_q3R CNV melt
9 nTc GAPDH B2 CNV melt
10 4 q2R_q2F US specific
11 4 P3F_q3R US specific
12 4 GAPDH B2 US specific
13 Blank q2R_q2F US specific
14 Blank P3F_q3R US specific
15 Blank GAPDH B2 US specific
16 nTc q2R_q2F US specific
17 nTc P3F_q3R US specific
18 nTc GAPDH B2 US specific
19 2 q1F_q1R CNV melt
20 2 GAPDH B2 CNV melt
21 4 q2F_P2R CNV melt
22 4 GAPDH B2 CNV melt
23 Blank q1F_q1R CNV melt
24 Blank q2F_P2R CNV melt
25 Blank GAPDH B2 CNV melt
26 nTc q1F_q1R CNV melt
27 nTc q2F_P2R CNV melt
28 nTc GAPDH B2 CNV melt

Notes:

  • Opaque white plates
  • Final reaction volumes = 15 μL
  • Each reaction had 3.0 μL of 750 nM F/R primers, 20 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 (water-only for nTc)
    • 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


Run PCR - Bio-Rad CFX96

  • 95°C, 3 min
  • 45 x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec]
  • 72°C, 30 sec
  • Melt: 60°C -- +0.2°C/ 5 sec --> 95°C


Results

  • Hypothesis 1 - CNV melt: CRISPR editing will result in a lower loading-normalized peak height value, compared to untreated cells
  • Hypothesis 2 - US-specific amplification: CRISPR editing will result in a lower loading-normalized Cq value, compared to untreated cells

CRISPR 1 - CNV melt

  • Primer pair 1 (q2F_q3R)
    • Untreated control Peak_q2F_q3R / Peak_GAPDH =
    • CRISPR treated Peak_q2F_q3R / Peak_GAPDH =
    • Conclusion:
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 1 - CNV melt

  • Primer pair 2 (P3F_q3R)
    • Untreated control Peak_P3F_q3R / Peak_GAPDH =
    • CRISPR treated Peak_P3F_q3R / Peak_GAPDH =
    • Conclusion:
    • Notes: some signal from nTc, not-so-good primer pair

CRISPR 2 - CNV melt

  • Primer pair (q1F_q1R)
    • Untreated control Peak_q1F_q1R / Peak_GAPDH =
    • CRISPR treated Peak_q1F_q1R / Peak_GAPDH =
    • Conclusion:
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 4 - CNV melt

  • Primer pair (q2F_P2R)
    • Untreated control Peak_q2F_P2R / Peak_GAPDH =
    • CRISPR treated Peak_q2F_P2R / Peak_GAPDH =
    • Conclusion:
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 4 - US-specific

  • Primer pair 1 (q2R_q2F)
    • Untreated control Cq_q2R_q2F / Cq_GAPDH =
    • CRISPR treated Cq_q2R_q2F / Cq_GAPDH =
    • Conclusion:
    • Notes: no signal from nTc, so good primer pair with no primer dimer signal

CRISPR 4 - US-specific

  • Primer pair 2 (P3F_q3R)
    • Untreated control Cq_P3F_q3R / Cq_GAPDH =
    • CRISPR treated Cq_P3F_q3R| / Cq_GAPDH =
    • Conclusion:
    • Notes: some signal from nTc, not-so-good primer pair


Summary Table

Experiment Method Norm Expt. value Norm control Conclusion
"1" - gRNA1/Cas9 primer pair 1 CNV melt ### ### ###
"1" - gRNA1/Cas9 primer pair 2 CNV melt ### ### ###



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