Etchevers:Notebook/STRA6 in eye development/2009/04/28

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 * style="background-color: #EEE"|[[Image:C14.jpg|128px]] Genetics of human eye development
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New ChIP protocol – tested with Sadaf and Nicolas
'''9 a.m. Prepare bead+antibody mix. '''

We removed 3x 50 μL of the Dynal/Invitrogen Protein-A magnetic beads, and then realized that 50 μL was already supposed to be enough for four IPs. Replaced. Continued with 50 μL even if it was only supposed to be for 3 antibodies.

Suspend beads and transfer to 0.5 mL low-adherence DNase-free microcentrifuge tubes. With some experience now, prefer the Eppendorf SafeLock variety as they seem to resist the later, hot incubations more consistently. 1.5 mL tubes make for poor movement of these volumes in rotation, and they stay either stuck in the lid or in the tip of tube.

Place on magnetic stand and remove supernatant.

Rinse with 500 μL RIPA (Upstate’s “Dilution”) buffer, removing tubes from rack and resuspending beads.

Repeat.

Remove supernatant and resuspend in 35 μL RIPA buffer (for 3 tubes).

Used 90 μL dilution buffer + 10 μL of rinsed beads and 4 μg each antibody – CHD7, or rabbit non-specific IgG, or 0.1 μg H3K4me3 antibody (Abcam) since that apparently works well. Place on rotating rack at 4ºC for 3 hours.

9:30 a.m. Prepare the chromatin on ice at all times!

Pre-cooled the agate mortar and pestle in the -70ºC freezer. Brought it upstairs with the C15 R700 embryo, on dry ice. Using a sterile pipette tip, knocked whole, flat embryo into the mortar and reduced to gross powder – not so small as when isolating RNA, as you don’t want to free all the nucleic acids just yet.

Prepare at least 1.2 mL PBS + 1x protease inhibitors (tablets, Roche). Also prepare some PBS + 10x protease inhibitors to keep on ice, to add to the SDS lysis buffer later (will need to bring up to room temperature to get the SDS back in solution).

Add 0.5 mL of the PBS to the frozen tissue, and thaw. Aspirate and place in a 1.5 mL centrifuge tube.

Add 13.5 μL 37% formaldehyde, vortex a few seconds and let fix for exactly 20 minutes. Mix gently on vortex every 5 minutes.

Add 50 μL 1M glycine and mix, leave 5 minutes at room temperature.

Spin down the pellet at 470g (2500 rpm on our centrifuge) at 4ºC, with soft deceleration.

Remove supernatant, resuspend in 0.5 mL cold PBS + protease inhibitors. Repeat spin and decant.

On ice, add to the pellet 120 μL of room temperature SDS Lysis Buffer with Roche protease inhibitors. Pipette to completely resuspend and leave on ice five minutes (go to the sonicator).

Sonicate 30 seconds at 30W power (Bronson, fine tip).

Centrifuge the lysate at full speed (>11000 rpm) for 10 minutes, 4ºC.

Transfer 100 μL supernatant into an iced tube labeled “chromatin”.

Add 30 μL of SDS lysis buffer to the pellet again, vortex, then centrifuge again full speed (>11000 rpm) for 10 minutes, 4ºC.

Recover another 50 μL supernatant to add to the chromatin tube. Avoid any visible pieces.

Sonicate this chromatin on ice 4x 30 seconds at 30W, leaving at least 1 minute between pulses.

Add 400 μL Dilution Buffer with Roche protease inhibitors and centrifuge full speed (>11000 rpm) for 10 minutes, 4ºC.

Remove 500 μL supernatant, transfer it to a new, iced tube labeled “sonicated chromatin”. Add 500 μL Dilution Buffer to the pellet again (which has 50 μL remaining, in theory), vortex and centrifuge at full speed (>11000 rpm) for 10 minutes, 4ºC.

Transfer the supernatant carefully to the “sonicated chromatin” tube with the first and mix. Remove 50 μL for determination of the total input to each IP (“total input” fraction). We put 150 μL into each IP. On the 30th, 3x150 μL; on May 4th, 4x 150 μL (although I am in doubt about this, and believe we actually only used 100 μL per fraction the second time around. This means that the total recovered DNA is not the same as Sadaf calculated on May 7th.)
 * Heather 16:59, 10 May 2009 (EDT): 'Note to myself - write Sadaf to revise the calculated yields to 83% of former results on the 2nd series from 4 May, as she did'' use only 125 rather than 150 μL - cf. this entry.

11:30 a.m. Immunoprecipitation

At around 12 noon, placed the antibody-bead complex tubes on the magnetic rack. Remove supernatant and replace with 150 μL chromatin in each of the CHD7, IgG and H3(K4me3) tubes.

Place in slow rotation at 4ºC for three hours in the cold room.

3 p.m. Rinses and DNA recovery

Brief centrifuge spin to bring down liquid, then place tubes on the magnetic rack. Remove supernatant and reserve until end, in case aspirated beads.

Resuspend rapidly in 150-200 μL cold Dilution buffer. Transfer beads to new 0.5 mL tubes to avoid carrying over non-specific DNA.

Resuspend using low-adherence plastic pipette tips, and place in cold room on rotator for 4 minutes.

Place tubes on magnetic rack, decant supernatant, and resuspend again in 150 μL cold Dilution Buffer. Place in cold room on rotator for 4 minutes.

Place tubes on magnetic rack, decant supernatant, and resuspend again in 150 μL Upstate’s Low Salt Buffer (or equivalent). Place in cold room on rotator for 4 minutes.

Place tubes on magnetic rack, decant supernatant, and resuspend again in 150 μL cold Upstate’s High Salt Buffer (or equivalent). Place in cold room on rotator for 4 minutes.

Place tubes on magnetic rack, decant supernatant, and resuspend again in 150 μL cold Upstate’s LiCl2 Buffer (or equivalent). Place in cold room on rotator for 4 minutes.

Place tubes on magnetic rack, decant supernatant, and resuspend again in 200 μL cold Tris-EDTA Buffer. Place in cold room on rotator for 4 minutes.

Make Complete Elution Buffer. Regular Elution buffer is: 60 μL Tris 1M pH 7.5 (final 20 mM) + 30 μL EDTA 0.5M (final 5 mM) + 30 μL NaCl 5M (final 50 mM) qsp 3 mL with ddH2O. Divide into 2x 1.5 mL and to the Complete EB tube, add 75 μL SDS 20% (final 1%) and 10 μL of proteinase K at 50 ng/μL (we used 20 mg/mL I believe).

Place tubes on magnetic rack, decant supernatant, and resuspend again in 200 μL cold Tris-EDTA Buffer. Transfer to new microcentrifuge 0.5 mL tubes. Place on magnetic rack and decant again,

Add to each tube (away from rack) 150 μL Complete Elution Buffer. To the 50 μL reserved Total Input fraction, add 10 μL NaCl 5M and 2 μL proteinase K.

Let the DNA dissociate from the beads for 3 hours at 67ºC on a rotating rack. Make sure the lids are well sealed!

7 p.m. DNA recovery

Place the tubes on the magnetic rack.

Transfer 150 μL supernatant to labeled CHD7/IgG/H3 IP tubes. It would have been more efficient for these to be 5 or 15 mL tubes, but they happened to be 1.5 mL tubes.

Add another 150 μL Elution Buffer (not the complete version) to the beads, resuspend, and we left them to rotate at room temperature overnight. Placed first supernatants at 4ºC.

More in next day's entry.
 * Heather 16:28, 10 May 2009 (EDT):


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