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
RPA-ssDNA pull down experiments D.C. 8-13-06
I. Bind biotin-oligo to streptavidin-agarose beads.
Wash streptavidin-agarose beads 2x in TE. For the following protocol you need 40ul of packed beads. Resuspend washed beads with 40ul of TE. This makes a total of 80ul of a 1:1 ratio of beads + TE.
Add 4ul of 100pmol/ul oligo (ours is 69b.p.) to beads and mix.
Incubate for 30minutes at room temperature and mix every 5 minutes.
Wash beads with binding buffer two times and resuspend with 40ul of binding buffer.
II. Bind RPA to DNA-Beads
Thaw RPA and spin in microfuge for 2 minutes at 4 degrees to pellet any precipitate.
For each pull-down you will need 2ul of packed DNA-beads (4ul of 1:1 slurry). This is equivalent to 20pmol of DNA.
For each reaction Add a four fold excess of RPA (80pmol) + an equal volume of binding buffer. You can do this in bulk (ie use 20ul of beads + 800pmol of RPA if you will be doing 10 pull downs.)
Also set up a negative control that does not have any RPA added. Just mix beads with binding buffer.
Incubate on rotator for 45minutes at room temperature.
The volume of beads per pull down is very small (2ul) and very difficult to see. So after the 45minute incubation I add a little bit of protein A agarose beads so that the volume of beads is larger. This doesnt seem to increase the background.
Wash beads three times with binding buffer.
The beads are now ready to do the pull-down.
III. Pull down
Lyse cells in NETN buffer (0.5%NP40 and 150mM salt) or CHAPS buffer (0.5-0.75%).
Clear lysate and measure protein concentration.
Set up two pull downs for each. One with the RPA-DNA-Beads and one with the DNA-Beads without any RPA. The amount of lysate to use depends on what you are looking for. When we did HA-ATRIP that had been transfected into 293T cells we used between 350ug and 700ug of lysate per reaction.
Incubate beads + lysate for 1.5-2hours at four degrees. I like to use the small 0.5ml eppendorf tubes. Rotate. Wash the beads three times with the lysis buffer. Add 20ul of 2X sample buffer. Load gel:
- 20ug of lysate
- DNA-beads pull down
- RPA-DNA beads pull down
Binding Buffer
10mM Tris pH=7.5
100mM NaCl
10% glycerol
0.02% IgePal
10 ug/ml BSA (Use the NEB BSA 100mg/ml stock)
0.05ug/ml aprotinin
0.05ug/ml leupeptin