User:Karmella Haynes/Notebook/Polycomb project/2010/12/01

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12/01/10

  • ✓ ChIP: pull-down; elute for DNA purification



ChIP co-IP optimization
> Samples:

  1. FTRx dx + α-H3K27me3 (07-449)
  2. FTRx dx + rabbit α-myc
  3. FTRx fx + α-H3K27me3 (07-449)
  4. FTRx fx + rabbit α-myc


> Buffers:

  1. Complete Buffer III
  2. Low Salt Immune Complex Buffer
  3. High Salt Wash Buffer
  4. LiCl Immune Complex Wash Buffer


> Bind beads (Note: *H3K27me3 ab 07-449 was Protein A purified, Millipore)

  • Pellet Protein A* beads slurry, wash 3x with Complete Buffer III, and resuspend in original volume (store remainder at 4°C).
  • Bind protein-ab complexes with 20 μL beads; rotate at 4°C/ 5 hours


Washes (Casey's protocol)

> Low Salt Immune Complex Buffer (LSIC) wash (2x, on ice)

  • Pellet beads at 3000 rpm/ 4 °C/ 2 min.

(Note: beads+complexes can be kept on ice for 1-2 hours)

  • Gently resuspend beads in 500 μL LSIC. Transfer to a fresh tube. Keep old tip inside original tube.
  • Incubate beads/LSIC on ice/ 2 min. (flick once during incubation).
  • Pellet beads at 3000 rpm/ RT/ 1 min.
  • With a fresh tip, add 500 μL LSIC to old tube w/ old tip. Use old tip to transfer LSIC to pellet (to collect up all remaining beads)
  • Repeat the previous wash and spin.


> High Salt Buffer (HSB) wash (1x, on ice)

  • Gently resuspend beads in 1 mL HSB.
  • Incubate beads/HSB on ice/ 2 min. (flick once during incubation).
  • Pellet beads at 3000 rpm/ RT/ 1 min.


> LiCl Immune Complex Buffer (LICB) wash (1x on ice)

  • Gently resuspend beads in 1 mL LICB.
  • Incubate beads/LICB on ice/ 2 min. (flick once during incubation).
  • Pellet beads at 3000 rpm/ RT/ 1 min.


> TE buffer wash (1x, RT)

  • Gently resuspend beads in 1 mL TE pH 7.5.
  • Incubate beads/TE @ RT/ 2 min. (flick once during incubation).
  • Pellet beads at 3000 rpm/ RT/ 30 sec.


Elution (Qingqing's protocol)

> Elute for DNA purification

  • Resuspend beads in 500 ul 1% SDS/TE buffer.
  • Heat at 65°C/ 15 min., vortex every 2 min.
  • Pellet beads, at 5000 rpm/ RT/ 3 min.
  • Transfer eluted chromatin (sup.) to a new 1.5 ml tube


DNA prep: day 1 (Qingqing's protocol)

  • Thaw 100 μL input sample. Add 400 μL 1% SDS/TE.
  • Add 20 μL Pronase (20 μg) to each ChIP and Input sample.
  • Incubate at 42°C/ 1 hr.
  • Incubate at 65°C/ 48 hrs