Haynes Lab:Notebook/CRISPR Editing/2014/08/07: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;"> Project name</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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* CRISPR on chromatin: transfections of g21, g22, g27 into Gal4EED/luc cells | * CRISPR on chromatin: transfections of g21, g22, g27 into Gal4EED/luc cells | ||
* Luc-mCh swap: building the donor sequence | * Luc-mCh swap: building the donor sequence | ||
* SURVEYOR | * SURVEYOR assay - C-on-C and mCherry | ||
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---- | ---- | ||
'''SURVEYOR assay for | '''SURVEYOR assay'''<br> | ||
Assistance from Week 2 Team 2 | |||
'''Planning''' | |||
{| {{table}} | |||
|- | |||
| Target || Genomic DNA (template) || Primers || Amplicon (bp) || Expected S-Cut fragments | |||
|- | |||
| mCherry gRNA1 || CRISPR 1d || qPCR 1F / P3R || 329 || ~229, 100 | |||
|- | |||
| mCherry gRNA2 || CRISPR 2d || " || 329 || ~52, 277 | |||
|- | |||
| mCherry gRNA4 || CRISPR 4d || " || 329 || ~180, 149 | |||
|- | |||
| 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 || " || 1295 || ~165, 1130 | |||
|- | |||
| 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 | |||
|} | |||
* mCherry annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit | |||
* Luciferase annotated map at https://benchling.com/rene/2014-cshl-synbio-mammalian-parts/FzI-puas-tk-luc-crispr-v-chromatin/edit | |||
'''PCR reaction''' | |||
* Reactions: 20 ng template DNA, 1x HF Phusion buffer, 0.2 mM dNTPs, 2.5 μL of 10 μM for each primer, 0.5 μL Phusion polymerase; in 50 μL final volume | |||
* Program | |||
** 98°C/ 30 sec | |||
** 30x [98°C/ 10 sec, 58°C/ 15 sec, 72°C/ 15 sec] | |||
** 72°C/ 10 min | |||
** 4°C hold | |||
'''PCR product clean-up''' | |||
* Used QIAquick PCR Purification kit | |||
* Elute with 50 μL H<sub>2</sub>O | |||
{| {{table}} | |||
|- | |||
| Sample || OD 260 || 260/280 || ng/uL | |||
|- | |||
| CRISPR 1d || 1.562 || 1.81 || 78.1 | |||
|- | |||
| CRISPR 2d || 0.273 || 1.71 || 14.0 | |||
|- | |||
| 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''' (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 | |||
# CRISPR 1d | |||
# CRISPR 2d | |||
# CRISPR 4d | |||
# g20 +dox | |||
# g20 -dox | |||
# g29 +dox | |||
# g29 -dox | |||
# +dox | |||
# -dox | |||
# U2OS mock | |||
* Thermal cycler program | |||
** 95ºC/ 10 min | |||
** 95ºC -- (‐2.0 ºC/s) --> 85ºC | |||
** 85ºC/ 1 min | |||
** 85ºC -- (‐0.3 ºC/s) --> 75ºC | |||
** 75ºC/ 1 min | |||
** 75ºC -- (‐0.3 ºC/s) --> 65ºC | |||
** 65ºC/ 1 min | |||
** 65ºC -- (‐0.3 ºC/s) --> 55ºC | |||
** 55ºC/ 1 min | |||
** 55ºC -- (‐0.3 ºC/s) --> 45ºC | |||
** 45ºC/ 1 min | |||
** 45ºC -- (‐0.3 ºC/s) --> 35ºC | |||
** 35ºC/ 1 min | |||
** 35ºC -- (‐0.3 ºC/s) --> 25 ºC | |||
** 25ºC/ 1 min | |||
** 4 ºC/ hold ∞ | |||
'''SURVEYOR nuclease reaction''' | |||
Latest revision as of 00:10, 27 September 2017
Project name | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
08/07/2014
CRISPR on chromatin: transfections
Luc-mCh swap: building the donor sequence
SURVEYOR assay
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