Haynes Lab:Notebook/CRISPR Editing/2014/07/08: Difference between revisions
Rene M Davis (talk | contribs) (Autocreate 2014/07/08 Entry for Haynes_Lab:Notebook/CRISPR_Editing) |
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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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==07//2014== | ==07/08/2014== | ||
Retrying the ligation, won't leave overnight again. | |||
<br> | |||
Annealing gRNA oligos<br> | |||
3ul 100uM top oligo<br> | |||
3ul 100uM bottom oligo<br> | |||
2ul 10x annealing buffer<br> | |||
12ul H2O<br> | |||
20 ul reactions <br><br> | |||
Assemblies<br> | |||
boil and cool method<br> | |||
Aluminum foil lid on the beaker to prevent condensation<br> | |||
Bring ~900 mL water to a boil in a large beaker (on a hot plate). Use a thermometer to check the temperature (>100°C).<br> | |||
Float the oligo mixtures in the boiling water for 10 min. Cover the beaker with aluminum foil to keep the air above the tube warm.<br> | |||
Turn off the heat source and allow the aluminum foil-covered water bath to slowly cool to room temperature (25°C) for several hours.<br><br> | |||
Ligate | |||
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| align="center" style="background:#f0f0f0;"|'''Label''' | |||
| align="center" style="background:#f0f0f0;"|'''Backbone''' | |||
| align="center" style="background:#f0f0f0;"|'''Insert''' | |||
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<br><br> | |||
Left for 1 hour, long transformation | |||
<br><br><br> | |||
Plated 100,000 cells into each well of two 6-well plates. Added G418 to final concentrations of 0, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, and 1500 μg/mL. | |||
<br>This is the second attempt because the first time G418 didn't kill the cells. We believe the drug was bad because it was expired by about 2 years. | |||
Latest revision as of 00:05, 27 September 2017
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07/08/2014Retrying the ligation, won't leave overnight again.
3ul 100uM top oligo 20 ul reactions
Left for 1 hour, long transformation
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