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
Nascent ssDNA by BrdU staining download
Nascent ssDNA by native BrdU staining (Jami, 2014)
Plate 300,000 U2OS cells in 6-well format (on either 15CIR or 22CIR coverslips) the afternoon before.
Cell Treatment:
- Pulse cells for 10 minutes with 10uM BrdU.
- Add HU to 3mM and ATRi to 5uM for 4h.
- After 4h, place cells on ice and proceed with immunostaining.
Immunostaining Procedure:
- Rinse cells once in PBS. Aspirate.
- Add .5-1mL Triton X solution, 10 min on ice.
- Wash 3x 1ml PBS carefully.
- Add 1-2mL paraformaldehyde/sucrose fixative, 10 minutes, room temp.
- Rinse 3x 1ml PBS. Cells can be stored in cold room/fridge at this step, leave in PBS.
- Block with 5% BSA/PBS, 15 min, room temp.
- Secure parafilm on lid of 6-well plate (if in coverslip format).
- Spin primary antibodies at 16,100g for 5 minutes to remove debris. (Not necessary)
- Add 35uL (50 for 22CIR) primary antibody as a drop on parafilm. Place coverslip face down on parafilm. 1h at 37deg.
- Rinse 3x 1ml PBS.
- Repeat 8-10 for secondary antibody. 30 minutes at room temp (dark).
- Thaw Prolong Gold with DAPI solution. Dab a small droplet using 1mL pipet tip onto slides. (5-10ul for 15CIR)
- Aspirate PBS from back of coverslip, aspirate all large water droplets from coverslip surface. Mount, remove bubbles and excess mounting solution. Allow slides to dry for a few hours or overnight before viewing.
Note: if doing 96-well format, add 35-50uL antibody solution to each well. Other steps should be similar.
Solutions:
Fixative (3% paraformaldehyde/2% sucrose): Dissolve 15g paraformaldehyde in 250mL water. Incubate 20min in 65-deg bath. Add 3 drops 10.0N NaOH. Incubate 5-10 minutes longer at 65deg. Add 50mL 10x PBS, 10g sucrose, dissolve and bring to 500mL. Filter, aliquot, and store at -20deg. Do not freeze-thaw, use a fresh tube each time.
Triton X-100 solution: (for 50mL) 0.5% Triton X-100 (1.25mL 20% stock), 20mM HEPES, pH 7.4 (2mL .5M stock), 50mM NaCl (.5mL 5M stock), 3mM MgCl2 (.15mL 1M stock), 300mM sucrose (5.134g). Dissolve, filter, store 4deg. This solution only lasts a few weeks.
Antibodies:
- Mouse anti-BrdU (BD, use 1:50 in 3% BSA/PBS)
- Goat anti-mouse Alexa 488 (Invitrogen, use 1:500 in 3% BSA/PBS)