Haynes Lab:Notebook/CRISPR Editing/2015/08/25: Difference between revisions

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Transfected siRNAs EZH2 and SUZ12 into gal4-EEDs with and without 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>
Resuspended duplex siRNAs with 100ul of unopened, RNase-free water for final concentration 20uM. Used RNase-away to reduce risk of degradation.
<br>
''Mix optimem and siRNA''
:11ul siRNA + 176ul Optimem in 1.5mL tube<br>
''Mix oligofectamine and optimem
:14.25ul oligofectamine + 57ul optimem
:let stand for 10 minutes<br>
''Mix DNA and oligofectamine''
:add 33ul oligofectamine to tubes with DNA
:let stand for 30 minutes
''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>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Component'''
| align="center" style="background:#f0f0f0;"|'''Final vol / well'''
|-
| 20uM siRNA duplex||2.5
|-
| Optimem||46
|-
| Oligofectamine||1.5
|-
| Total||50
|}
<br>
After 4 hours, add 125ul DMEM + 3x serum to each well.




Revision as of 13:44, 25 August 2015

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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

' 1 well 19 wells
ug g034 0.5 9.50
ul g034
ul water

Step 2: make DNA + optimem mixes

' 1 well 19 x
g034 39 add 741ul optimem

Step 3: add plus reagent to DNA+ opti

' 1 well
g034 add 19ul plus reagent

step 4: Make optimem + lipo mastermix

' 1 well for 18 wells (x 19.2)
optimem 47 902.4
lipo 3 57.6

Step 5: mix dna mix and lipo mix

' 1 well x 19
g034 50 add 950 lipo mix

Step 6: add lipo mix to cells
add 100ul of mix to each well


Transfected siRNAs EZH2 and SUZ12 into gal4-EEDs with and without dox
Cells plated yesterday looked good.

Cells plated at 30% were at about 80%
Cells plated at 10% and 20% were at about 50%

Transfected 2 wells for each siRNA and each cell type

Resuspended duplex siRNAs with 100ul of unopened, RNase-free water for final concentration 20uM. Used RNase-away to reduce risk of degradation.
Mix optimem and siRNA

11ul siRNA + 176ul Optimem in 1.5mL tube

Mix oligofectamine and optimem

14.25ul oligofectamine + 57ul optimem
let stand for 10 minutes

Mix DNA and oligofectamine

add 33ul oligofectamine to tubes with DNA
let stand for 30 minutes

While complexes form, prep cells

Removed media
Wash in 100ul PBS
Aspirate PBS
Add 200ul of plain DMEM

Add complexes to cells

Add 50ul complexes dropwise to each well


Component Final vol / well
20uM siRNA duplex 2.5
Optimem 46
Oligofectamine 1.5
Total 50


After 4 hours, add 125ul DMEM + 3x serum to each well.



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