Haynes Lab:Notebook/CRISPR Editing/2014/08/07: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 10: | Line 10: | ||
* 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 | ||
Line 38: | Line 38: | ||
---- | ---- | ||
'''SURVEYOR assay for | '''SURVEYOR assay'''<br> | ||
Assistance from Week 2 Team 2 | |||
PCR primers | |||
{| | |||
|- | |||
| Target || Genomic DNA (template) || Primers || Amplicon | |||
| mCherry gRNA1d || CRISPR 1d || q1F / P3R || 329 bp | |||
|} | |||
* Annotated map at https://benchling.com/cshlsynbio/mammalian-crispr/Rc6-kah154/edit | |||
'''PCR templates''' | |||
# CRISPR "1" | |||
# CRISPR "2" | |||
# CRISPR "4" | |||
# "Blank" (3x) | |||
'''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 "1" || --- || 1.82 || 40.3 | |||
|- | |||
| CRISPR "2" || --- || 1.62 || 4.7 | |||
|- | |||
| CRISPR "4" || --- || 1.85 || 9.8 | |||
|- | |||
| Blank 1 || --- || 1.80 || 22.9 | |||
|- | |||
| Blank 2 || --- || 1.54 || 5.0 | |||
|- | |||
| Blank 3 || --- || 1.85|| 21.5 | |||
|} | |||
* Combined Blank 1 and Blank 3, ~22 ng/μL in 100 uL volume | |||
'''Mismatch annealing''' | |||
* Testing 2 methods | |||
** IDT approach - mix the mutant PCR sample with a wild type reference sample to ensure heteroduplex formation | |||
** 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 15 μL | |||
# 100 ng CRISPR "1" + 100 ng Blank (IDT approach) | |||
# 200 ng CRISPR "1" (Zhang approach) | |||
# 200 ng Blank (wild type control) | |||
* 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 ∞ | |||
Revision as of 09:09, 7 August 2014
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||
08/07/2014
CRISPR on chromatin: transfections
Luc-mCh swap: building the donor sequence
SURVEYOR assay PCR primers
|