Protocols
- Protocols
- Competent Cells Protocol
- hTERT-RPE serum starvation for synchronization
- Immunofluorescence Protocol
- hTERT-RPE serum starvation for synchronization
- Flow Cytometry Manuals
- Fork regression/restoration/footprinting
- SMARCAL1 DNA binding ATPase assays
- PI and BrdU Flow Cytometry Protocols
- Rong’s 293T suspension+mass spec
- Lysis with DNAse for all nuclear proteins including histones
- DNA Combing Protocol
- DNA fiber labeling
- Comet Assay
- Nascent ssDNA IF assay
- 53BP1 foci
- Retro/Lentivirus production
- PEI transfection of 293T cells
- siRNA transfection optimization
- Flag-His purification mammalian cells
- ATRflox cells Cre
- ATR kinase assay
- G2/M checkpoint assay
- Freezing/Thawing cells in Liquid Nitrogen
- RPA-ssDNA pull down
- GST protein purification from bacteria
- Bacterial expression/His purification
- HeLa siRNA/plasmid transfection
- Protein solubility test in E.coli
siRNA Transfection Optimization - 11.02.09
DAY 1
Preparation of siRNA plates
Dilute siRNAs to final concentration of 0.5pmol/ul = 0.5micomolar in 1x siRNA dilution buffer. Prepare 0.5ml total.
All Stars Neg Control siRNA - Qiagen cat# 1027281
All Stars Hs Cell Death siRNA – Qiagen cat# 1027299
10nM siRNA Concentration = 1.25pmol = 2.5ul of siRNA/well
Distribute siRNAs in a 96 well plate.
Aliquot : 52.5ml siRNA/well; Rows A-H; Column 1.*
Add 210 ml OptiMEM to each well containing siRNA.*
Pipette to mix.
*These volumes are for plating 2 – 96 well plates and 10 columns of siRNA.
See siRNA Plating Master Plate Template.
Use this “master mix” plate to aliquot 12.5ml/well siRNA/OptiMEM into 2 96 well plates using a multichannel pipettor.
See Experiment 11.02.09 Template for siRNA plating.
Store plates covered at room temperature until needed.
Preparation of Cells
Trypsinize and count cells. Prepare 20ml of cells at a concentration of 1.280 x 105 cells/ml in growth media in a 50ml conical tube. This is 2.56x106 cells total.
Perform 1.6x serial dilution. Transfer 12.5ml of cells into new 50ml conical and add 7.5ml of growth media. Repeat. See diagram below.
cells/ml 128000 80000 50000 31250
#1 is for rows G and H of the lipid/RNAi plates
#2 is for rows E and F of the lipid/RNAi plates
#3 is for rows C and D of the lipid/RNAi plates
#4 is for rows A and B of the lipid/RNAi plates
Store cells: 37 degrees; 5%CO2; until ready to use.
Preparation of lipid complexes
HiPerfect
Prepare 3 Hiperfect mixes.
- 240ul Optimem + 10ul Hiperfect – Column 1 (0.5ul/well)
- 235ul Optimem + 15ul Hiperfect – Column 2 (0.75ul/well)
- 230ul Optimem + 20ul Hiperfect – Column 3 (1.0ul/well)
Dharmafect 1
Prepare 3 Dharmafect mixes.
- 249ul Optimem + 1ul Dharmafect1 – Column 4 (0.05ul/well)
- 248ul Optimem + 2ul Dharmafect1 – Column 5 (0.1ul/well)
- 246ul Optimem + 4ul Dharmafect1 – Column 6 (0.2ul/well)
Lipofectamine RNAiMax
Prepare 3 Lipofectamine RNAiMax mixes
- 248ul Optimem + 2ul RNAiMax – Column 7 (0.1ul/well)
- 246ul Optimem + 4ul RNAiMax – Column 8 (0.2ul/well)
- 244ul Optimem + 6ul RNAiMax – Column 9 (0.3ul/well)
See Lipid Plating Master Plate Template.
Combine Lipid Mixes + siRNA
Use the multichannel pipettor to aliquot 12.5ul into each of the siRNA plates. Mix by pipetting up and down. Incubate for 20minutes
See Experiment 11.02.09 Template.
Combine Lipid Mixes + siRNA + Cells
Dispense cells into Lipid+siRNA plates by first placing the cell suspension into a reservoir. Using the multichannel pipettor add 100ul per well to the appropriate row of the plate.
See Cell Plating Template.
DAY 2
Split Cells
Each well of a 96 well plate is split into four wells in a 384 well plate. Total cell/media volume - 40ml/well. Trypsinize cells: 20 ml/well.
Media 160 ml/well.
DAY 5 and/or Day 6
Added alamar Blue: 4ml/well.
Incubated: 37°; 5% CO2; 5 hours.
Read SpectraMax5 HTS Lab.
