Haynes Lab:Notebook/CRISPR Editing/2014/08/07: Difference between revisions
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(10 intermediate revisions by the same user not shown) | |||
Line 52: | Line 52: | ||
| mCherry gRNA2 || CRISPR 2d || " || 329 || ~52, 277 | | mCherry gRNA2 || CRISPR 2d || " || 329 || ~52, 277 | ||
|- | |- | ||
| mCherry gRNA4 || CRISPR | | mCherry gRNA4 || CRISPR 4d || " || 329 || ~180, 149 | ||
|- | |- | ||
| mCherry || | | mCherry || mock || " || 329 || no cutting | ||
|- | |- | ||
| luciferase gRNA20 || g20 || 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 | ||
|- | |- | ||
| luciferase | | luciferase gRNA20 || g20 -dox || " || 1295 || ~165, 1130 | ||
|- | |- | ||
| luciferase || mock || " || 1295 || no cutting | | luciferase gRNA29 || g29 +dox || " || 1295 || ~395, 900 | ||
|- | |||
| luciferase gRNA29 || g29 -dox || " || 1295 || ~395, 900 | |||
|- | |||
| luciferase || mock +dox || " || 1295 || no cutting | |||
|- | |||
| luciferase || mock -dox || " || 1295 || no cutting | |||
|} | |} | ||
Line 84: | Line 90: | ||
| Sample || OD 260 || 260/280 || ng/uL | | Sample || OD 260 || 260/280 || ng/uL | ||
|- | |- | ||
| CRISPR | | CRISPR 1d || 1.562 || 1.81 || 78.1 | ||
|- | |- | ||
| CRISPR | | CRISPR 2d || 0.273 || 1.71 || 14.0 | ||
|- | |- | ||
| CRISPR | | CRISPR 4d || 1.77 || 1.588 || 79.3 | ||
|- | |- | ||
| | | g20 +dox || 1.408 || 1.79 || 70.4 | ||
|- | |- | ||
| | | g20 -dox || 1.057 || 1.78 || 52.9 | ||
|- | |- | ||
| | | g29 +dox || 0.987 || 1.77 || 49.4 | ||
|- | |||
| g29 -dox || 0.856 || 1.82 || 42.8 | |||
|- | |||
| +dox || 1.300 || 1.77 || 65.0 | |||
|- | |||
| -dox || 1.169 || 1.8 || 58.5 | |||
|- | |||
| U2OS mock || --- || --- || not done yet | |||
|} | |} | ||
'''Mismatch annealing''' | '''Mismatch annealing''' (continued on 08/07/14) | ||
* 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 | |||
# | # CRISPR 1d | ||
# | # CRISPR 2d | ||
# | # CRISPR 4d | ||
# g20 +dox | |||
# g20 -dox | |||
# g29 +dox | |||
# g29 -dox | |||
# +dox | |||
# -dox | |||
# U2OS mock | |||
Line 129: | Line 146: | ||
** 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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