Haynes Lab:Notebook/CRISPR Editing/2015/07/28: 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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** P145/146 (...@255) = 100 bp; Tm = 57, 58.8°C | ** P145/146 (...@255) = 100 bp; Tm = 57, 58.8°C | ||
* Number of unique reactions: 8 x 3 = 24 reactions | * Number of unique reactions: 8 x 3 = 24 reactions | ||
* Number of wells needed: 24 x 3 = '''72 wells''' (1 plate) | * Number of wells needed: 24 rxns x 3 reps = '''72 wells''' (1 plate) | ||
Primer master mixes | Primer master mixes | ||
* Multiplier = 8 x 3 + 3 = 27 | * Multiplier = 8 rxns x 3 reps + 3 = 27 | ||
# P147/148 | # P147/148 | ||
# P143/144 | # P143/144 | ||
# P145/146 | # P145/146 | ||
Note: reference primers (GAPDH B2) are not here yet | |||
{| {{table}} | {| {{table}} | ||
|- | |- | ||
| Reagent || Vol. || (x27) | | Reagent || Vol. μL || (x27) | ||
|- | |- | ||
| 750 nM F/R primer mix || | | 750 nM F/R primer mix || 3.0 || 81.0 | ||
|- | |- | ||
| 2x SYBR (Roche) || | | 2x SYBR (Roche) || 7.5 || 202.5 | ||
|- | |||
| || 10.5 || 283.5 | |||
|} | |||
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 | |||
# CRISPR 1d (135.9) | |||
# CRISPR 2d (204.9) | |||
# CRISPR 3d (11.3) | |||
# CRISPR 4d (145.7) | |||
# donor Stop@72 & 1:10 dilution (120.6 & 17.8) | |||
# donor Stop@162 & 1:10 dilution (95.1 & 12.1) | |||
# donor Stop@255 & 1:10 dilution (83.2 & 10.2) | |||
# no template control (water) | |||
{| {{table}} | |||
|- | |||
| Reagent || Vol. μL || (x12) | |||
|- | |||
| 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 | |||
* 95°C, 3 min | |||
* 40x [95°C, 10 sec / 57°C, 10 sec / 72°C, 10 sec (measure)] | |||
* 72°C, 30 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
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