Haynes Lab:Notebook/CRISPR Editing/2015/06/27: Difference between revisions
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Rene M Davis (talk | contribs) (Autocreate 2015/06/27 Entry for Haynes_Lab:Notebook/CRISPR_Editing) |
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== | ==06/27/2015== | ||
transfected gal4-eeds with puro and dox with four dilutions of g034 in triplicate. did the following twice, once for each drug treatment<br><br> | |||
'''Step 1: Diluting the DNA''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''ug of DNA for 3.2 wells''' | |||
| align="center" style="background:#f0f0f0;"|'''ul plasmid''' | |||
| align="center" style="background:#f0f0f0;"|'''water (32ul total)''' | |||
|- | |||
| g034 0.25||0.8|||| | |||
|- | |||
| g034 0.5||1.6|||| | |||
|- | |||
| g034 0.75||2.4|||| | |||
|- | |||
| g034 1.0||3.2|||| | |||
|} | |||
<br> | |||
'''Step 2: make DNA + optimem mixes''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 well''' | |||
| align="center" style="background:#f0f0f0;"|'''3.2 x''' | |||
|- | |||
| g034 0.25||39||add 124.8ul optimem | |||
|- | |||
| g034 0.5||39||add 124.8ul optimem | |||
|- | |||
| g034 0.75||39||add 124.8ul optimem | |||
|- | |||
| g034 1.0||39||add 124.8ul optimem | |||
|} | |||
<br> | |||
'''Step 3: add plus reagent to DNA+ opti''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 well''' | |||
| align="center" style="background:#f0f0f0;"|'''3.2 x''' | |||
|- | |||
| g034 0.25||1||add 3.2ul plus reagent | |||
|- | |||
| g034 0.5||1||add 3.2ul plus reagent | |||
|- | |||
| g034 0.75||1||add 3.2ul plus reagent | |||
|- | |||
| g034 1.0||1||add 3.2ul plus reagent | |||
|} | |||
<br> | |||
'''Step 4: Make optimem + lipo mastermix''' | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 well''' | |||
| align="center" style="background:#f0f0f0;"|'''for 12 wells (x 13.2)''' | |||
|- | |||
| optimem||47||620.4 | |||
|- | |||
| lipo||3||39.6 | |||
|} | |||
<br> | |||
'''Step 5: mix dna mix and lipo mix''' | |||
{| {{table}} | |||
| g034 0.25||add 160 lipo mix | |||
|- | |||
| g034 0.5||add 160 lipo mix | |||
|- | |||
| g034 0.75||add 160 lipo mix | |||
|- | |||
| g034 1.0||add 160 lipo mix | |||
|} | |||
'''Step 6: add 100ul to each well''' | |||
<br><br> | |||
Collected transfected luc14 cells for flow and gDNA extraction: | |||
<br> | |||
:aspirate media | |||
:add 200ul PBS, rock plate, aspirate PBS | |||
:add 100ul trypsin, let sit for a few minutes, tap plate to unstick cells | |||
:add 400ul media, pipette to collect cells, move to 1.5mL microcentrifuge tube | |||
:remove tubes from hood | |||
:spin in benchtop centrifuge at 0.2x1000RCF for 3 minutes | |||
:remove media with P1000 | |||
:resuspend in 400ul cold PBS | |||
:move 200ul to 2mL tube for flow, use remaining 200ul for gDNA extraction | |||
<br> | |||
<!-- ##### 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. ##### --> |
Revision as of 18:32, 30 June 2015
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06/27/2015transfected gal4-eeds with puro and dox with four dilutions of g034 in triplicate. did the following twice, once for each drug treatment
Step 6: add 100ul to each well
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