Haynes Lab:Notebook/CRISPR Editing/2014/08/06: Difference between revisions
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* Genomic DNA extractions | * Genomic DNA extractions | ||
* qPCR: CRISPR on chromatin & mCherry (single plate) | |||
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* U2OS = KAH154 CRISPR cells | * U2OS = KAH154 CRISPR cells | ||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''OD 260''' | |||
| align="center" style="background:#f0f0f0;"|'''260/280''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/uL''' | |||
| align="center" style="background:#f0f0f0;"|'''Average ng/uL''' | |||
|- | |||
| Genomic DNA 7 (gRNA1d)||1.523||1.9||76.1||76.0 | |||
|- | |||
| Genomic DNA 7 (gRNA1d)||1.514||1.88||75.7|| | |||
|- | |||
| Genomic DNA 7 (gRNA1d)||1.526||1.89||76.3|| | |||
|- | |||
| Genomic DNA 8 (gRNA2d)||0.315||1.79||15.7||15.6 | |||
|- | |||
| Genomic DNA 8 (gRNA2d)||0.321||1.74||16.1|| | |||
|- | |||
| Genomic DNA 8 (gRNA2d)||0.302||1.75||15.1|| | |||
|- | |||
| Genomic DNA 9 (gRNA4d)||2.092||1.94||104.6||104.4 | |||
|- | |||
| Genomic DNA 9 (gRNA4d)||2.083||1.94||104.2|| | |||
|- | |||
| Genomic DNA 9 (gRNA4d)||2.087||1.93||104.4|| | |||
|- | |||
| Genomic DNA 10 (-g)||1.889||1.94||94.5||94.0 | |||
|- | |||
| Genomic DNA 10 (-g)||1.882||1.93||94.1|| | |||
|- | |||
| Genomic DNA 10 (-g)||1.87||1.94||93.5|| | |||
|- | |||
| Genomic DNA 1||0.772||1.93||38.6||39.0 | |||
|- | |||
| Genomic DNA 1||0.782||1.89||39.1|| | |||
|- | |||
| Genomic DNA 1||0.784||1.83||39.2|| | |||
|- | |||
| Genomic DNA 2||2.17||1.96||108.5||108.2 | |||
|- | |||
| Genomic DNA 2||2.178||1.95||108.9|| | |||
|- | |||
| Genomic DNA 2||2.146||1.95||107.3|| | |||
|- | |||
| Genomic DNA 3||1.016||1.89||50.8||50.6 | |||
|- | |||
| Genomic DNA 3||1.02||1.9||51|| | |||
|- | |||
| Genomic DNA 3||1.001||1.94||50.1|| | |||
|- | |||
| Genomic DNA 4||1.398||1.91||69.9||68.7 | |||
|- | |||
| Genomic DNA 4||1.35||1.93||67.5|| | |||
|- | |||
| Genomic DNA 4||1.376||1.9||68.8|| | |||
|- | |||
| Genomic DNA 5||4.144||1.99||207.2||206.7 | |||
|- | |||
| Genomic DNA 5||4.179||2||209|| | |||
|- | |||
| Genomic DNA 5||4.08||2||204|| | |||
|- | |||
| Genomic DNA 6||4.907||1.99||245.4||244.0 | |||
|- | |||
| Genomic DNA 6||4.9||1.98||245|| | |||
|- | |||
| Genomic DNA 6||4.829||1.99||241.5|| | |||
|} | |||
---- | |||
'''qPCR: CRISPR on chromatin & mCherry''' | |||
'''qPCR Master Reaction list''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Reaction''' | |||
| align="center" style="background:#f0f0f0;"|'''Templates''' | |||
| align="center" style="background:#f0f0f0;"|'''Primers (Targets)''' | |||
| align="center" style="background:#f0f0f0;"|'''Amplicon''' | |||
| align="center" style="background:#f0f0f0;"|'''Method''' | |||
|- | |||
| 1||g20 +dox||g020US-F/ g020US-R||152||CNV | |||
|- | |||
| 2||g20 +dox||GAPDH B2||||CNV | |||
|- | |||
| 3||g20 -dox||g020US-F/ g020US-R||152||CNV | |||
|- | |||
| 4||g20 -dox||GAPDH B2||||CNV | |||
|- | |||
| 5||g29 +dox||g029US-F/ g029US-R||141||CNV | |||
|- | |||
| 6||g29 +dox||GAPDH B2||||CNV | |||
|- | |||
| 7||g29 -dox||g029US-F/ g029US-R||141||CNV | |||
|- | |||
| 8||g29 -dox||GAPDH B2||||CNV | |||
|- | |||
| 9||+dox||g020US-F/ g020US-R||152||CNV | |||
|- | |||
| 10||+dox||g029US-F/ g029US-R||141||CNV | |||
|- | |||
| 11||+dox||GAPDH B2||||CNV | |||
|- | |||
| 12||-dox||g020US-F/ g020US-R||152||CNV | |||
|- | |||
| 13||-dox||g029US-F/ g029US-R||141||CNV | |||
|- | |||
| 14||-dox||GAPDH B2||||CNV | |||
|- | |||
| 15||1d||q2F / q3R||187||CNV | |||
|- | |||
| 16||1d||GAPDH B2||||CNV | |||
|- | |||
| 17||2d||q1F / q1R||142||CNV | |||
|- | |||
| 18||2d||GAPDH B2||||CNV | |||
|- | |||
| 19||4d||q2F / P2R||140||CNV | |||
|- | |||
| 20||4d||GAPDH B2||||CNV | |||
|- | |||
| 21||mock||q2F / q3R||187||CNV | |||
|- | |||
| 22||mock||q1F / q1R||142||CNV | |||
|- | |||
| 23||mock||q2F / P2R||140||CNV | |||
|- | |||
| 24||mock||GAPDH B2||||CNV | |||
|} | |||
* 72 wells | |||
'''Primer Master Mixes''' | |||
# g020US-F/ g020US-R (12 wells) | |||
# g029US-F/ g029US-R (12) | |||
# q2F / q3R (6) | |||
# q1F / q1R (6) | |||
# q2F / P2R (6) | |||
# GAPDH B2 (30) | |||
'''Template Master Mixes''' | |||
# g20+dox (6 wells) | |||
# g20-dox (6) | |||
# g29+dox (6) | |||
# g29-dox (6) | |||
# +dox (9) | |||
# -dox (9) | |||
# 1d (6) | |||
# 2d (6) | |||
# 4d (6) | |||
# mock (12) | |||
Notes: | |||
* Opaque white plates | |||
* Final reaction volumes = 14 μL | |||
* Each reaction had 3.0 μL of 750 nM F/R primers, 50 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 | |||
* previous nTc for g020US-F/ g020US-R, g029US-F/ g029US-R, q2F / q3R, q1F / q1R, q2F / P2R, and GAPDH B2 confirmed no background signal under identical conditions | |||
'''Run PCR - Bio-Rad CFX96''' | |||
* 95°C, 3 min | |||
* '''40x''' [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)] | |||
* 72°C, 30 sec | |||
* Melt: 70°C -- '''+0.1°C'''/ 5 sec (measure) --> 95°C | |||
Revision as of 08:24, 7 August 2014
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08/06/2014
Genomic DNA extractions Samples
qPCR: CRISPR on chromatin & mCherry
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