siRNA library screen of mouse ES cells
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Optimize cell number for experiment.
Optimize TOP transfection reagent and Luciferase assay conditions.
Optimize siRNA transfection using GAPDH control kit and various transfection reagents.
Optimize plate reader for luciferase assay conditions and plate size.
Order all reagents with the possibility of repeating a minimum of 4 times.
Order enough sterile filter tips and white TC plates – Very important to use filter tips.
CGR8 ES cells: siRNA transfection using 0.2 l Dharmafect1 per well with 10 nM siRNA.
TOP transfection using 0.2 g TOP plasmid and 0.5 L Lipofectamine 2000.
 Catalogue #: Alumaseal II sterile seals for 96 well plates (for aliquot of library): 351006024 –
Fisher $50/50 seals.
Axygen full skirt 96 well PCR plate (for aliquot of library): 10011-228 – VWR
$84.54/case of 50.
White 96 well TC plates: 07200722 Corning 3362 – Fisher $384/case of 100.
KDAlert GAPDH Enzyme Assay: AM1639 – Applied Biosystems $268/375
rxns.
Lipofectamine 2000: 11668-019 – Invitrogen $580/1.5 ml.
Dharmafect1: T2001-03 – Dharmacon $415/1.5 ml.
Luciferin: L6882 – Sigma $1190/50 mg.
Coenzyme A: C3144 – Sigma $231/100 mg.
Optimem and GMEM media
1.
Bring up ES cells, passage 2-3 times.
2.
Subculture and dilute to 3000 cells/100 L in complete ES media.
3.
Calculate how many plates to use for screen X2. Gelatin coat 96 well plates for 10 minutes with 100
L, remove by flicking into sink. For example, the kinase library had 7 pooled siRNA 96 well plates
and the phosphatase library had 3 plates for a total of 10 plates per cell line per repetition – 40 plates
in all (two repetitions and two cell lines). 40 plates are too much to handle at one time, high risk for
contamination, so maybe do one repetition instead.
4.
Plate 100 L of cell suspension into each well, incubate overnight.
5.
Prepare for siRNA transfection. Use aliquoted library that has been pooled and is at a
concentration of 200 nM. Calculate 10 l of siRNA per well per repetition. Do two repetitions.
Transfer siRNA to clean 96 well PCR plates. Prepare Dharmafect transfection reagent. Calculate
0.2 L Dharmafect1 + 14.8 l Optimem per well (15 L total). Add 15 l Dharmafect/Optimem
to 10 l siRNA per well per repetition. Incubate 30 minutes.
6.
Remove media from 96 well plates by flicking into sink. Add new complete ES media to wells, 75
L per well.
7.
Aliquot using multipipetor, 25 L of siRNA/Dharmafect/Optimem to each well. Remember to
label carefully. Incubate for 48 hours.
8.
Prepare mastermix of TOP reporter plasmid plus Lipofectamine 2000. Calculate 0.2 g of TOP
DNA per well + 25 L of Optimem per well, multiplied by the number of wells that need to
be transfected. Prepare 0.5 l Lipofectamine + 25 L Optimem, multiplied by the number of wells
that need to be transfected. Combine the two solutions and incubate for 30 minutes. Change media
on cells; remove by flicking into the sink. Add 100 L Optimem to each well with the multipipetor.
Add 50 L of DNA+Lipo+Optimem per well using multipipetor. Incubate 24 hours.
9.
Harvest with fresh NP-40 Lysis buffer, 20 L per well. NP-40 lysis buffer: 10 mL 1 M Tris pH 7.8, 5
mL 10% NP-40, 85 mL ddH2O, fresh 50 mM DTT (2 mL buffer + 1 L 1M DTT).
10.
Luciferase assay of cell lysates in plate. Calculate how much you will need to add 100 L per well.
For 20 plates, you will need 300 mL.
Luciferase Assay Buffer: (Promega)
Stock:
9 ml
6 ml
3 ml
final conc.
500 mM Tricine
360 µl
240 µl
120 µl
20 mM Tricine
100 mM Mg-carbonate 97
64
32
1.07 mM MgCO3
1 M MgSO4
24
16
7.5
2.67 mM MgSO4
50 mM EDTA
18
12
6
0.1 mM EDTA
1 M DTT
299
198
99
33.3 mM DTT
10 mM coenzymeA
243
162
81
270 µM coenzymeA
100 mM luciferin
42.3
28.2
14
470 µM luciferin
100 mM ATP
47.7
31.8
16.5
530 µM ATP
DDH2O
7869
5248
2624
Buffer has to be room temperature before using.
Aliquot 100 L of Luciferase Assay Buffer per well using multipipetor and read in plate reader
immediately. Plate reader setting should have been optimized prior to the experiment.