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
Do siRNA transfection 3 days before experiment. You will need 1 well of a 6 well dish at about 50-90% confluent for each sample.
Damage cells. Wait some time. The do staining. If using IR or UV, I recommend waiting for 1 or 2 hours. A time course would be nice to do. Use 5-8Gy of IR or 30J/m2 of UV. You can also do with HU 1mM. I’m not sure what time point with HU is best but maybe 2-6 hours is where I would start.
Staining of cells:
Harvest cells as if for western blot but be sure to harvest both floating and adherant cells. Then resuspend in 300ul of cold PBS. Add 700ul of cold 100% ethanol and mix immediately to fix cells. Wait at least 60 minutes for fixation to work. Place cells on ice or in –20 during fixation.
Wash cells 2x with cold PBS. All centrifugaton should be at 1300 xg for 2 minutes.
The first wash you do not have to resuspend cell pellet. 2nd wash resuspend pellet by pipetting gently.
Add 1.0ml of ice cold 0.25% tritionX-100 in PBS to permeabalize. Do on ice for 15 minutes. Centrifuge then rinse cells (without pipetting) in PBS/1%BSA. Centrifuge again and resuspend cells in 100ul of room temp PBS/1%BSA + anti-PhosphH3 antibody (use at 1:75 dilution). Incubate for 1 hour at room temperature. I usually resuspend cells every 15 minutes by flicking tube.
Wash 2x with PBS/1%BSA. I resuspend 1x and just rinse 1x.
Resuspend cells in 100ul of PBS/1%BSA + anti-rabbit FITC antibody at 1:50 dilution. Incubate 30 minutes R.T. in dark.
Wash 1x with PBS/1%BSA and 1x with PBS
Stain with 25ug/ml propidium iodide+0.1mg/ml rnase in 1ml of PBS for 30 minutes at room temperature.
Measure on flow cytometer immediately. Maybe can store cells overnight at 4 degrees if necessary.