Haynes Lab:Notebook/CRISPR Editing/2014/08/07: Difference between revisions
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(3 intermediate revisions by the same user not shown) | |||
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| mCherry gRNA4 || CRISPR 4d || " || 329 || ~180, 149 | | mCherry gRNA4 || CRISPR 4d || " || 329 || ~180, 149 | ||
|- | |- | ||
| mCherry || | | mCherry || mock || " || 329 || no cutting | ||
|- | |- | ||
| luciferase gRNA20 || g20 +dox || gRNA 021 US P F / gRNA 027 ES P R || 1295 || ~165, 1130 | | luciferase gRNA20 || g20 +dox || gRNA 021 US P F / gRNA 027 ES P R || 1295 || ~165, 1130 | ||
Line 108: | Line 108: | ||
| -dox || 1.169 || 1.8 || 58.5 | | -dox || 1.169 || 1.8 || 58.5 | ||
|- | |- | ||
| | | U2OS mock || --- || --- || not done yet | ||
|} | |} | ||
'''Mismatch annealing''' (continued on 08/07/14) | |||
'''Mismatch annealing''' | |||
* Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway | * Zhang Lab approach - do not mix with a wild type reference, since ~50% or more of the amplicons in the mutated cell sample will be wild type anyway | ||
* 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 20 μL | ** 200 ng clean PCR, 1x HF Phusion buffer, in final volume of 20 μL | ||
# CRISPR 1d | # CRISPR 1d | ||
Line 124: | Line 121: | ||
# g20 +dox | # g20 +dox | ||
# g20 -dox | # g20 -dox | ||
# g29 +dox | # g29 +dox | ||
# g29 -dox | |||
# +dox | |||
# -dox | |||
# U2OS mock | |||
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** 4 ºC/ hold ∞ | ** 4 ºC/ hold ∞ | ||
'''SURVEYOR nuclease reaction''' | |||
Revision as of 07:32, 8 August 2014
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08/07/2014
CRISPR on chromatin: transfections
Luc-mCh swap: building the donor sequence
SURVEYOR assay
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