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
2-10-12
Modified by D. Cortez 1/2022
PEI Transfection
Cells: 293T
Volume: 10 cm plate
Day 1: Split 293T cells into a 10cm plate with complete DMEM so that they are roughly 50% confluent. Higher than 50% will decrease transfection efficiency. 2.5-3.0 million cells per 10cm dish the day before works well according to Nancy.
Day2: Add 4 ug total DNA to 100 uL of Optimem in 1.5 mL tube.
Add 24 uL of PEI.
Mix by vortexing.
Leave at room temperature for 10-15 minutes.
Aspirate media off cells and replace with 10 mL of fresh complete DMEM with 10% FBS
Add PEI/DNA mixture dropwise while swirling the plate of 293T cells.
Incubate overnight at 37°C.
Day 3: Split into 15cm2 plate
Day 4: Collect or wait one more day – one extra day usually helps increase yield.
Day 5: Collect if you did not collect on Day 4.
PEI is linear polyethylenimine (MW 25,000) from Polyscience, Inc. (Cat # 23966). Make a 1 mg/mL solution of PEI in water, neutralize to pH 7.0 with HCl and filter sterilize. Use a hot plate and stir bar to dissolve PEI; it may take 6 – 8 hours to dissolve. It is stable at 4°C for at least 3 months. Keep bulk of aliquots stored at -80°C.
Note: It is also possible to do the PEI transfection on the same day as plating the cells (even without allowing them to attach). In that case, double the number of cells utilized.