Haynes Lab:Notebook/CRISPR Editing/2015/08/25

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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 g0340.59.50
ul g034
ul water

Step 2: make DNA + optimem mixes

' 1 well 19 x
g03439add 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)
optimem47902.4
lipo357.6

Step 5: mix dna mix and lipo mix

' 1 well x 19
g034 50add 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 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

For siRNA complexes

' per well 3.1
20uM siRNA duplex2.57.75
Optimem46142.6
Oligofectamine1.54.65
Total50155

For blank transfections

' per well 3.1
water2.57.75
Optimem46142.6
Oligofectamine1.54.65
Total50155

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

Mix oligofectamine and optimem

19.8ul oligofectamine + 79.2ul optimem
let stand for 10 minutes

Mix DNA and oligofectamine

add 23.25ul oligofectamine to tubes with RNA or water
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



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



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