Haynes Lab:Notebook/CRISPR Editing/2014/07/08: 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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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> | 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> | 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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Label''' | |||
| align="center" style="background:#f0f0f0;"|'''Backbone''' | |||
| align="center" style="background:#f0f0f0;"|'''Insert''' | |||
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| 1||pX330||g14 | |||
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| 2||pX330||g5 | |||
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| 3||pX330||g13 | |||
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| 4||pX330||gn1T | |||
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| 5||pX330||gn1B | |||
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| 6||pX330||gn3T | |||
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| 7||pX330||gnBb B | |||
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| Bb 5||pX335||bb ctrl | |||
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| 8||pX335||gn3T | |||
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| 9||pX335||gn1B | |||
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| 10||pX335||gn1T | |||
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| 11||pX335||gn3bB | |||
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Left for 1 hour, long transformation | |||
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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
Project name | Main project page Previous entry Next entry | ||||||||||||||||||||||||||||||||||||||||||
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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