Haynes Lab:Notebook/CRISPR Editing/2014/08/05: Difference between revisions
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# CRISPR "2" | # CRISPR "2" | ||
# CRISPR "4" | # CRISPR "4" | ||
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'''qPCR melt curve optimization - mCherry CRISPR 1''' | |||
<br>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) | |||
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'''Run PCR - Bio-Rad CFX96''' | '''Run PCR - Bio-Rad CFX96''' | ||
* 95°C, 3 min | * 95°C, 3 min | ||
* | * '''30x''' [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec] | ||
* 72°C, 30 sec | * 72°C, 30 sec | ||
* Melt: 60°C -- +0. | * Melt: 60°C -- '''+0.1°C'''/ 5 sec --> 95°C | ||
Revision as of 08:22, 5 August 2014
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08/05/14
SURVEYOR assay PCR primers [Rene, please add this info]
PCR templates
qPCR melt curve optimization - mCherry CRISPR 1
Optimization parameters
Notes:
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