Work Flow Plan for siRNA Library Screen – Mouse Kinase Library with Mouse ES Cells.
Library contains 1713 siRNA for 571 genes. Using pooled Library. I will need 10 plates per cell line per
repetition. Total plates: 20 for CGR8, 20 for G45.
Prepare Work Flow Plan for the experiment. Do search for conditions that you can start with.
1.
Optimize transfection.
-cell number.
3000-5000 cells per well.
-transfection reagent.
Will use Lipofectamine and Dharmafect. Potentially could use Dharmafect for siRNA first
then Lipo for reporter. 0.2 ul Dharmafect + 14.8 ul mediaFBS + 10 ul diluted siRNA. 0.5 ul
Lipo + 25 ul Optimem combined with 0.2 ug DNA + 25 ul Optimem pipeted onto cells
with 50 ul Optimem in well already.
-standard vs reverse transfection.
ES cells do not like reverse transfection with lipofectamine, but may be able to do
reverse with Dharmafect.
-length of transfection.
O/N with media change in the AM, grow for 24-48 hours.
2.
Optimize plate reader
-luminescence vs ultrasensitive luminescence.
I use Lum for ES cells.
-time of plate reading.
currently at 1s but could go down to 0.2s.
-stability of luciferase.
TopFlash is less stable so will need to do faster readings.
-inject one well then read if stability is a problem.
3.
Optimize siRNA transfection using GAPDH readout (KDAlert kit from Applied Biosystems)
-cell number.
3000-5000
-transfection reagent.
Dharmafect is good for siRNA but not DNA.
-standard vs reverse transfection.
could use Dharmafect with reverse on day 1 and Top with Lipo on day 3 with assay on
day 4.
-time period.
3 days is optimal for knockdown.
-type of siRNA and concentration.
silencer at 20 nM is standard.
4.
Optimize DNA transfection and siRNA transfection together
-cotransfect DNA and siRNA, grow 3 days
-transfect siRNA on Day 1, then on Day 3, transfect reporter plasmid with assay on Day 4
5.
Order reagents and supplies.
-white plates.
Corning is the cheapest, with Perkin Elmer also supplys white plates with clear
bottoms.
-transfection reagents.
Lipofectamine and Dharmafect.
-cell culture reagents.
GMEM, FCS, Optimem.
-assay specific reagents.
Luciferin, coenzyme A – need at least 100 ml.
-tips.
Perkin Elmer if using robot.
6.
Decide when to start screen
-manual aliquoting vs robot.
The robot has not been optimized yet and will take some time to change media etc if
doing multiple transfections on separate days. Manual may be faster with multipipetor.
-pooled vs single siRNA.
May want to start with pooled siRNA and then do single if you see multiple differences.
-size of experiment that you are able to do at one time (~40 plates)
Optimal is doing the whole library in two replicates at one time but this may not be
feasible. Separate into two duplicates on separate days if need be.
-prepare library – dilute stocks with ddH2O.
Make your own stocks of the library at 200 nM with enough for 3-4 replicates.
-screen is done in two replicates (100 plates).
-maxiprep of reporter plasmid (1200 ug needed).