Haynes Lab:Notebook/CRISPR Editing/2015/08/25: 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. ##### --> | ||
== | ==08/25/2015== | ||
'''Transfect gal4+puro and gal4+dox with g034, collect at 24, 48, and 72 hours''' | |||
Step 1: Making .5ug/10ul plasmid stocks | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 well''' | |||
| align="center" style="background:#f0f0f0;"|'''19 wells''' | |||
|- | |||
| ug g034||0.5||9.50 | |||
|- | |||
| ul g034|||| | |||
|- | |||
| ul water|||| | |||
|} | |||
Step 2: make DNA + optimem mixes | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 well''' | |||
| align="center" style="background:#f0f0f0;"|'''19 x''' | |||
|- | |||
| g034||39||add 741ul optimem | |||
|} | |||
Step 3: add plus reagent to DNA+ opti | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 well''' | |||
|- | |||
| g034 ||add 19ul plus reagent | |||
|} | |||
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 18 wells (x 19.2)''' | |||
|- | |||
| optimem||47||902.4 | |||
|- | |||
| lipo||3||57.6 | |||
|} | |||
Step 5: mix dna mix and lipo mix | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''1 well''' | |||
| align="center" style="background:#f0f0f0;"|'''x 19''' | |||
|- | |||
| g034 ||50||add 950 lipo mix | |||
|} | |||
Step 6: add lipo mix to cells<br> | |||
add 100ul of mix to each well | |||
<br><br> | |||
'''Transfected siRNAs EZH2 and SUZ12 into gal4-EEDs with dox'''<br> | |||
Cells plated yesterday looked good. | |||
:Cells plated at 30% were at about 80% | |||
:Cells plated at 10% and 20% were at about 50%<br> | |||
Transfected 2 wells for each siRNA and each cell type | |||
<br><br> | |||
For siRNA complexes | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''per well''' | |||
| align="center" style="background:#f0f0f0;"|'''3.1''' | |||
|- | |||
| 20uM siRNA duplex||2.5||7.75 | |||
|- | |||
| Optimem||46||142.6 | |||
|- | |||
| Oligofectamine||1.5||4.65 | |||
|- | |||
| Total||50||155 | |||
|} | |||
For blank transfections | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''per well''' | |||
| align="center" style="background:#f0f0f0;"|'''3.1''' | |||
|- | |||
| water||2.5||7.75 | |||
|- | |||
| Optimem||46||142.6 | |||
|- | |||
| Oligofectamine||1.5||4.65 | |||
|- | |||
| Total||50||155 | |||
|} | |||
''Mix optimem and siRNA or water'' | |||
:7.75ul each siRNA + 124ul Optimem in 1.5mL tube | |||
:7.75ul water + 124ul Optimem in 2 1.5mL tubes<br> | |||
''Mix oligofectamine and optimem | |||
:19.8ul oligofectamine + 79.2ul optimem | |||
:let stand for 10 minutes<br> | |||
''Mix DNA and oligofectamine'' | |||
:add 23.25ul oligofectamine to tubes with RNA or water | |||
:let stand for 30 minutes<br> | |||
''While complexes form, prep cells'' | |||
:Removed media | |||
:Wash in 100ul PBS | |||
:Aspirate PBS | |||
:Add 200ul of plain DMEM<br> | |||
''Add complexes to cells''<br> | |||
:Add 50ul complexes dropwise to each well | |||
<br> | |||
<br> | |||
After 4 hours, add 125ul DMEM + 3x serum to each well. | |||
Latest revision as of 01:05, 27 September 2017
Project name | Main project page Previous entry Next entry | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
08/25/2015Transfect gal4+puro and gal4+dox with g034, collect at 24, 48, and 72 hours Step 1: Making .5ug/10ul plasmid stocks
Step 2: make DNA + optimem mixes
Step 3: add plus reagent to DNA+ opti
step 4: Make optimem + lipo mastermix
Step 5: mix dna mix and lipo mix
Step 6: add lipo mix to cells
Transfected 2 wells for each siRNA and each cell type
For blank transfections
Mix optimem and siRNA or water
Mix oligofectamine and optimem
Mix DNA and oligofectamine
While complexes form, prep cells
Add complexes to cells
|