Haynes Lab:Notebook/CRISPR Editing/2015/05/27: 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. ##### --> | ||
== | ==05/27/2015== | ||
Redo qPCR with the right control primers, TBP<br> | |||
Dilutions for 2ng gDNA per well: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Tube label''' | |||
| align="center" style="background:#f0f0f0;"|'''Cell type''' | |||
| align="center" style="background:#f0f0f0;"|'''gRNA''' | |||
| align="center" style="background:#f0f0f0;"|'''plasmid 2''' | |||
| align="center" style="background:#f0f0f0;"|'''ug DNA''' | |||
| align="center" style="background:#f0f0f0;"|'''ul lipo''' | |||
| align="center" style="background:#f0f0f0;"|'''rep''' | |||
| align="center" style="background:#f0f0f0;"|'''ng/µL''' | |||
| align="center" style="background:#f0f0f0;"|'''dilute 1:20''' | |||
| align="center" style="background:#f0f0f0;"|'''ul dilution''' | |||
| align="center" style="background:#f0f0f0;"|'''ul water''' | |||
|- | |||
| 1||Gal4-EED||g034||-||0.5||3||1||81.058||4.05||1.73||14.02 | |||
|- | |||
| 2||Gal4-EED||g034||-||0.5||3||2||86.697||4.33||1.61||14.14 | |||
|- | |||
| 3||Gal4-EED||g034||-||0.5||5||1||23.89||1.19||5.86||9.89 | |||
|- | |||
| 4||Gal4-EED||g034||-||0.5||5||2||24.767||1.24||5.65||10.10 | |||
|- | |||
| 5||Gal4-EED||g034||-||1||3||1||85.32||4.27||1.64||14.11 | |||
|- | |||
| 6||Gal4-EED||g034||-||1||3||2||70.244||3.51||1.99||13.76 | |||
|- | |||
| 7||Gal4-EED||g034||-||1||5||1||13.387||0.67||10.46||5.29 | |||
|- | |||
| 8||Gal4-EED||g034||-||1||5||2||23.213||1.16||6.03||9.72 | |||
|- | |||
| 9||Luc14||g034||GFP||-||-||1||104.239||5.21||1.34||14.41 | |||
|- | |||
| 10||Luc14||g034||GFP||-||-||2||64.965||3.25||2.16||13.59 | |||
|- | |||
| 11||Luc14||g034||GFP||-||-||3||39.096||1.95||3.58||12.17 | |||
|- | |||
| 12||Gal4-EED+dox||g034||GFP||-||-||1||160||8||0.88||14.88 | |||
|- | |||
| 13||Gal4-EED+dox||g034||GFP||-||-||2||129||6.45||1.09||14.66 | |||
|- | |||
| 14||Gal4-EED+dox||g034||GFP||-||-||3||132||6.6||1.06||14.69 | |||
|- | |||
| 15||water||-||-||-||-||-||-||-||-||15.75 | |||
|} | |||
<br><br> | |||
Step 2: make primer master mixes. <br>Will have to add 3.5x to each triplicate gDNA tube so can calculate mastermixes from that<br> | |||
14 samples, 1 tube for no template control, total of 15 tubes per primer. | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''3.5/triplicate tube''' | |||
| align="center" style="background:#f0f0f0;"|'''15.7''' | |||
|- | |||
| primer||3||10.5||164.85 | |||
|- | |||
| SYBR||7.5||26.25||412.125 | |||
|- | |||
| Total||10.5||36.75||576.975 | |||
|} | |||
Make these mixes for each primer pair, add 36.75 to each triplicate tube, then pipette 15ul into 3 well of the plate. | |||
<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit below this line unless you know what you are doing. ##### --> |
Latest revision as of 00:59, 27 September 2017
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05/27/2015Redo qPCR with the right control primers, TBP
Make these mixes for each primer pair, add 36.75 to each triplicate tube, then pipette 15ul into 3 well of the plate. |