Haynes Lab:Notebook/CRISPR Editing/2015/07/28: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
(fix raw html notebook nav) |
|||
(6 intermediate revisions by one other user not shown) | |||
Line 2: | Line 2: | ||
|- | |- | ||
|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}}}}]]}} | ||
|- | |- | ||
| colspan="2"| | | colspan="2"| | ||
Line 51: | Line 51: | ||
Template master mixes | Template master mixes (Nanodrop reads, ng/uL) | ||
* Note: Donor DNA are column-cleaned PCR products; make dilutions in case the concentrations are too high | |||
* Multiplier = 3 rxns x 3 reps + 3 = 12 | * Multiplier = 3 rxns x 3 reps + 3 = 12 | ||
# CRISPR 1d | # CRISPR 1d (135.9) | ||
# CRISPR 2d | # CRISPR 2d (204.9) | ||
# CRISPR 3d | # CRISPR 3d (11.3) | ||
# CRISPR 4d | # CRISPR 4d (145.7) | ||
# donor Stop@72 | # donor Stop@72 & 1:10 dilution (120.6 & 17.8) | ||
# donor Stop@162 | # donor Stop@162 & 1:10 dilution (95.1 & 12.1) | ||
# donor Stop@255 | # donor Stop@255 & 1:10 dilution (83.2 & 10.2) | ||
# no template control (water) | # no template control (water) | ||
Line 66: | Line 67: | ||
| Reagent || Vol. μL || (x12) | | Reagent || Vol. μL || (x12) | ||
|- | |- | ||
| DNA ( | | DNA (20 ng) || 0.5 || 6.0 | ||
|- | |- | ||
| | | PCR H<sub>2</sub>O || 4.0 || 48.0 | ||
|- | |- | ||
| || | | || 4.5 || 54.0 | ||
|} | |} | ||
Loading | |||
* Template mix, 13.5 into each rep 1 well (A1, A4, A7...) | |||
* Primer mix, 31.5 into each rep 1 well (A1, B1, C1...H1...) | |||
PCR - Bio-Rad CFX96 | PCR - Bio-Rad CFX96 | ||
95°C, 3 min | * 95°C, 3 min | ||
* 40x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)] | |||
72°C, 30 sec | * 72°C, 30 sec | ||
Melt: 70°C -- +0.1°C/ 5 sec ( | * Melt: 70°C/ 5 sec -- +0.1°C (measure) --> 95°C/ 5 sec | ||
CONCLUSIONS | |||
* Overall, Ct values strangely very high for nTc and some templates (values of 10 - 24) | |||
* ??? - P147/148 - no signal from Donor stop 1 control | |||
* P143/144 - appears to work well | |||
TROUBLE SHOOTING | |||
* Use less DNA template | |||
Latest revision as of 01:04, 27 September 2017
Project name | Main project page Previous entry Next entry | ||||||||||||||||||||||||
07/28/2015
mCherry qPCR/melt pre-course test (at CSH)
Note: reference primers (GAPDH B2) are not here yet
Loading
PCR - Bio-Rad CFX96
TROUBLE SHOOTING
|