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

{| width="800"
 * style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

12/01/10

 * &#x2713; 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) (Note: beads+complexes can be kept on ice for 1-2 hours)
 * Pellet beads at 3000 rpm/ 4 °C/ 2 min.
 * 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


 * }