Test qPCR primers for ChIP
Dilute Gal4-EED 1 sonicated and column purified DNA from ChIP 1:100
- 1ul diluted template
- 1ul F primer
- 1ul R primer
- 7ul water
- 10ul Taq 2x MM
||Tm for taq
Plasmid: MV13-KAH60 6 297ng/ul, confirmed by Cass
- 1ul SgrAI
- 1ul Fastdigest buffer or Cutsmart
- 1ul XbaI
- 1ul Cutsmart buffer or Fastdigest
2ul plasmid, 10ul total rxn
Incubate at 37 for 1 hour
Confirm on gel
SgrAI destroyed the backbone. Need to cut with more vector, less enzyme and for a shorter time.
XbaI cuts in cutsmart, SgrAI cuts in FD buffer.
Western primary stain
Make or thaw more blocking buffer (5% BSA, 1%PBS, 1% tween, bring to volume with DI water from tap). Get blocked nitrocellulose membranes from 4deg room.
Make parafilm pouches! Cut a strip of parafilm that can be folded over to make a pouch just a few millimeters wider than each membrane. Lay the membrane in the unmade pouch and make the pouch around the membrane by smooshing double-folded parafilm edges with the spatula tool. leave the top open, pipette in 1mL blocking buffer into the pouch, set aside and watch for leaks. Make the other pouches the same way. Dilute the antibodies, 4ul each into 1mL blocking buffer. Confirm that none of the pouches have leaked. Add the antibodies to the appropriate pouches, LABEL each pouch with the antibody. Seal the tops, rotate at 4degC overnight.
- H3-ab1791 Rb pAb from abcam
- New - Millipore rabbit poly 07-449 (purchased this month)
- Old - Millipore rabbit poly 07-449 (purchased last year)