Haynes Lab:Notebook/CRISPR Editing/2014/09/21: 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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<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
== | ==09/21/2014== | ||
Ran the PCR for gRNA 20 samples from yesterday on a gel. Ran 3μL of each sample and 2μL and 1μL of ladder. Only the first three worked, even though the other two worked the other day. | |||
<br><br> | |||
Annealing all of each of gRNA 20, plus the PCRs from the other day that worked. Will just go through the protocol to see if I can even do Surveyor with the concentrations I'm getting. | |||
<br><br> | |||
Going to try more PCR. Will do gRNA 21 samples and all the Luc14 samples. If I can get all the Luc14 samples through the entire protocol, I can know if any of the gRNAs aren't cutting at all and won't have to keep trying the chromatin-induced samples. | |||
<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Set 1''' | |||
| align="center" style="background:#f0f0f0;"|'''Set 2''' | |||
|- | |||
| gRNA 21 + dox 1||gRNA 20 Luc 14 | |||
|- | |||
| gRNA 21 + dox 2||gRNA 21 Luc 14 | |||
|- | |||
| gRNA 21 - dox 1||gRNA 22 Luc 14 | |||
|- | |||
| gRNA 21 - dox 2||gRNA 27 Luc 14 | |||
|- | |||
| gRNA 21 Luc 14||gRNA 29 Luc 14 | |||
|} | |||
Master mix 1 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''10.5 rxns''' | |||
|- | |||
| H20||23.5||246.75 | |||
|- | |||
| 5x buffer||10||105 | |||
|- | |||
| dNTPs||1||10.5 | |||
|- | |||
| FP||2.5||26.25 | |||
|- | |||
| RP||2.5||26.25 | |||
|- | |||
| DMSO||1.5||15.75 | |||
|- | |||
| ||41|| | |||
|} | |||
add 41ul of MM 1 to each tube<br> | |||
add 4ul of each template to each tube (400ng each) | |||
Master mix 2 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''10.5 rxns''' | |||
|- | |||
| H20||4.5||47.25 | |||
|- | |||
| Phusion||0.5||5.25 | |||
|} | |||
add 5 ul of MM 2 to each tube | |||
<br> | |||
PCR <br> | |||
:98°C for 3 min | |||
:36 cycles | |||
::98°C for 10s | |||
::66°C for 30s | |||
::72°C for 60s | |||
:72°C for 10 min | |||
:4°C forever | |||
<br><br> | |||
transferred 40ul of the first three annealing reactions and 35ul of the last two to new tubes. added 1ul enhancer and 2ul of surveyor nuclease. incubate at 42deg C for 1 hour. | |||
<br> | |||
added 5ul of water to whatever is left in the PCR tubes from annealing. will run those as a no-nuclease control. | |||
<br> | |||
Ran Surveyor of gRNA 20 samples on 1% gel | |||
<br> | |||
Ran PCR products of gRNA 21 samples and all Luc 14 samples on gel. | |||
Annealed. | |||
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__NOTOC__ | __NOTOC__ |
Latest revision as of 00:19, 27 September 2017
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09/21/2014Ran the PCR for gRNA 20 samples from yesterday on a gel. Ran 3μL of each sample and 2μL and 1μL of ladder. Only the first three worked, even though the other two worked the other day.
Master mix 1
add 41ul of MM 1 to each tube
add 5 ul of MM 2 to each tube
Ran PCR products of gRNA 21 samples and all Luc 14 samples on gel. Annealed. |