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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| Genomic DNA 6||4.829||1.99||241.5|| | | Genomic DNA 6||4.829||1.99||241.5|| | ||
|} | |} | ||
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'''qPCR: CRISPR on chromatin & mCherry''' | |||
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| 24||mock||GAPDH B2||||CNV | | 24||mock||GAPDH B2||||CNV | ||
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* 72 wells | |||
'''Primer Master Mixes''' | '''Primer Master Mixes''' | ||
# g020US-F/ g020US-R ( | # g020US-F/ g020US-R (12 wells) | ||
# g029US-F/ g029US-R | # g029US-F/ g029US-R (12) | ||
# q2F / q3R | # q2F / q3R (6) | ||
# q1F / q1R | # q1F / q1R (6) | ||
# q2F / P2R | # q2F / P2R (6) | ||
# GAPDH B2 | # GAPDH B2 (30) | ||
'''Template Master Mixes''' | '''Template Master Mixes''' | ||
# g20+dox | # g20+dox (6 wells) | ||
# g20-dox | # g20-dox (6) | ||
# g29+dox | # g29+dox (6) | ||
# g29-dox | # g29-dox (6) | ||
# +dox | # +dox (9) | ||
# -dox | # -dox (9) | ||
# 1d | # 1d (6) | ||
# 2d | # 2d (6) | ||
# 4d | # 4d (6) | ||
# mock | # 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 | |||
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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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