Cumbers:Protocols

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(ChIP)
Line 11: Line 11:
*Dilute beads 1:1 in IP buffer, doubling each 20ul to 40ul.  Aliquot 40ul slurry to a clean eppendorf (make sure to add exactly the same amount each time, cut off pipette tip)
*Dilute beads 1:1 in IP buffer, doubling each 20ul to 40ul.  Aliquot 40ul slurry to a clean eppendorf (make sure to add exactly the same amount each time, cut off pipette tip)
-
*Now take the top 90% (so 180ul in my case) of your pre-cleared chromatin and add it to the 40ul beads you just prepared, avoid taking the very bottom of the chromatin, to avoid carrying over any precipitated material, which could cause poor quality results.
+
*Now take the top 90% (so 180ul in my case) of your pre-cleared-Ab-added-chromatin and add it to the 40ul beads you just prepared, avoid taking the very bottom of the chromatin, to avoid carrying over any precipitated material, which could cause poor quality results.
*Rotate tubes 4.c for 45 mins on a rotating platform (20-30 rotations per minute)
*Rotate tubes 4.c for 45 mins on a rotating platform (20-30 rotations per minute)

Revision as of 16:02, 19 January 2008

Fast ChIP

From Nelson et al, Nature 2006 "Protocol for the fast chromatin immunoprecipitation (ChIP) method

ChIP

  • prepare aliquots of 0.2 million cells/IP, (~200ul in my case)
  • Add Ab to pre-cleared chromatin samples, incubate in ultrasonic water bath for 15 mins (must be determined for each Ab used) at 4.c (in cold room). Could just do on a rotisserie if you don't have a ultrasonic water bath, but would take longer.
  • Do mock IP, without Ab, or pre-immune serum as control.
  • Spin down chromatin, 10 mins @ 12,000g
  • meanwhile.. wash 3x protein A beads (20 ul per IP sample) in IP buffer (see paper)
    • A wash is in 1ml IP buffer, spin centrifuge 2,000g for a few seconds.
  • Dilute beads 1:1 in IP buffer, doubling each 20ul to 40ul. Aliquot 40ul slurry to a clean eppendorf (make sure to add exactly the same amount each time, cut off pipette tip)
  • Now take the top 90% (so 180ul in my case) of your pre-cleared-Ab-added-chromatin and add it to the 40ul beads you just prepared, avoid taking the very bottom of the chromatin, to avoid carrying over any precipitated material, which could cause poor quality results.
  • Rotate tubes 4.c for 45 mins on a rotating platform (20-30 rotations per minute)
  • You just have a slower rotisserie, so rotate for longer (2hrs?) at 4.c
  • Centrifuge the slurry at 2k g for a few seconds at RT or 4.c. Remove supernatant and discard.
  • Wash the beads 5x (which now have the Ab-protein-of-interest stuck to it) 1ml cold IP buffer without inhibitors (why not?).

DNA isolation

  • Add 100ul 10% wt/vol Chelax 100, slurry direct to the washed beads
  • Vortex samples for 10 secs, boil for 10 mins, 95.c in pcr machine (or heat block)
  • To get your input DNA sample, take a aliquot of pre-cleared chromatin
Personal tools