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
Freezing cells:
- Start with healthy, growing cells. Typically use a 10cm2 dish that is nearly confluent to freeze one or two vials. For cells like HeLa and 293T that have a lot of cells per dish it is OK to freeze two vials per 10cm2 dish. Cells like normal fibroblasts or untransformed cells work better if you just freeze one vial.
- Prepare freezing media: Growth media with 20%FBS + 10%DMSO. Ice cold
- Prepare freezing vials: Label with the cell type, date, and your initials. Place on ice.
- Trypsinize cells, inactivate trypsin with growth media. Centrifuge cells 1000rpm 5minutes. Remove the growth media.
- Resuspend cell pellet in ice cold freezing media. Transfer cells to cold freezing vial on ice.
- Transfer freezing vial to -80 box. Incubate in -80 overnight.
- Transfer vials to Liquid Nitrogen after one night. If you are freezing a new cell type then enter into the excel spread sheet (DC_cell_lines_liquid nitrogen.xls). Each cell type is frozen in 1 row of cells. If you are freezing additional vials of cells of a cell line already frozen then please place the new vials in the same location as the old vials. Do not start a new box. Do not keep a box of your cells. Do not place in random locations that people cannot find.
Notes: We store our cells in the vapor phase of the liquid nitrogen. That means that we only place 10 boxes per rack so the bottom racks that are submerged in the liquid nitrogen do not have boxes in them.
Thawing cells:
- Pre-warm growth media.
- Remove vial from liquid nitrogen. Incubate in the water bath just until thawed. Transfer cells to a 15ml conical containing 10ml of growth media. Centrifuge 1000 rpm 5minutes. Remove the media. Resuspend in 10ml of fresh media and plate in 10cm2 dish.
Note: Most cell types should attach within the first 6hours. However, HCT116 cells (including the ATRflox cells) require up to 48 hours to attach.