6 protocols
AccessionType
image_acquisition
According to Manufacturer's instructions
labeling
DNA Precipitation Step 4 - Precipitation of DNA.
Add 150 µl of "proteinase K mix" to each sample.
Incubate for 2 hours at 37¡C in the warm room.
Extract 2 times with 1 volume of phenol (Sigma Cat. P-4557; OK to use at 4¡C).
Spin for about 5 minutes at room temperature for each extraction.
Extract once with 1 volume of chloroform/isoamyl alcohol (Sigma Cat.C-0549).
Add NaCl to 200 mM final (use 8 µl of 5 M stock for 200 µl of sample).
Add 2 volumes of cold EtOH and vortex briefly.
Incubate at -20¡C for at least 15 minutes.
Spin at 14,000 rpm for 10 minutes at 4¡C.
Pour off the supernatant, add 1 ml cold 70% EtOH, vortex briefly and spin at 14,000 rpm for 5 minutes at 4¡C.
Pour off the supernatant, spin briefly and remove the remaining liquid with a pipette.
Let the pellet dry for a couple of minutes and resuspend the pellet in 30 µl TE containing 10 µg RNaseA (add 33 µl of 10 mg/ml RNaseA to 1 ml of TE).
Incubate for 1 hour at 37¡C in the warm room.
Purify using Qiagen PCR purification kit.
Elute with 50 µl of 10 mM Tris pH 8.0.
Store at -20¡C or place on ice and proceed to step 5.
Stop at this stage if you are just going to do a gene-specific PCR, without hybridizing to glass slide arrays.

Ligation-mediated PCR
Step 6 - Ligation-mediated PCR.

Add 6 µl of 3M NaOAc (pH 5.2) to linker-ligated DNA.
Mix by vortexing and add 130 µl cold EtOH.
Mix by vortexing and spin for 15 minutes at 4¡C.
Pour off supernatant and wash with 500 µl 70% EtOH.
Spin for 5 minutes at 4¡C.
Pour off supernatant, spin and remove any remaining liquid with a pipette.
Resuspend in 25 µl ddH2O and place on ice.
Add 15 µl of PCR labeling mix:
4 µl 10X ThermoPol reaction buffer (NE Biolabs)
5.75 µl ddH2O
2 µl low T mix (5 mM each dATP, dCTP, dGTP; 2 mM dTTP)
2 µl Cy3-dUTP or Cy5-dUTP (use Cy5 for IP DNA and Cy3 for WCE DNA)
1.25 µl oligo oJW102 (40 µM stock)
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15 µl Total

Try to use Cy3 or Cy5 from the same batch i.e. avoid mixing batches.
Transfer to PCR tubes on ice, place in PCR machine and start program "Cy3" or Cy5" (the programs are stored under "Main" in our PCR machines or under "FR" in the tetrad PCR machine in the back room):
Step Time/Instruction Temp.
Notes 1 2 min 55¡C (make this longer if you have a lot of samples)
2 5 min 72¡C
3 2 min 95¡C
4 30 sec 95¡C
5 30 sec 55¡C
6 1 min 72¡C
7 go to step 4 for X* more times
8 4 min 72¡C
9 hold 4¡C
*32 cycles (total) for Cy5 and 34 cycles for Cy3
Add 10 µl of polymerase mix during step 1 of PCR:
8 µl ddH2O
1 µl 10X ThermoPol reaction buffer (NE Biolabs)
1 µl Taq polymerase (5 U/µl) (Perkin Elmer: Use Cat. #N801-0060 i.e. regular Taq., do not use AmpliTaq Gold)
0.01 µl PFU Turbo (2.5 U/µl) (Stratagene Cat #600250-51)
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10 µl Total

Run 5 µl on a 1.5% agarose gel. (The PCR product should be a smear ranging from 200 bp to 600 bp with an average size of 400 bp).
Purify with Qiaquick PCR purification kit.
Elute in 50 µl.
Add 6 µl 3M NaOAc, mix and add 130 µl cold EtOH.
Mix and spin for 15 minutes at 4¡C.
Pour off supernatant and wash with 500 µl of 70% EtOH.
Spin for 5 minutes at 4¡C.
Pour off supernatant, spin and remove any remaining liquid with a pipette.
Store PCR products at -20¡C.
Keep in a closed box to prevent exposure to light.
hybridization
Pre-hybridization, Probe Preparation, Hybridization and Wash

Step 7 - Pre-hybridization

Incubate slide in 3.5X SSC, 0.1% SDS, 10 mg/ml BSA for 20 minutes at room temperature with agitation (use a stir bar on setting "5") and then 20 minutes at 50¡C suing a pre-warmed solution (place Coplin jar in water bath; use a fresh solution).
Wash slide using RO water.
Blow-dry with nitrogen or by placing slides in a rack and spinning in a centrifuge for 2 min @ 1 krpm.

Step 7a - Probe preparation During slide pre-hybridization, resuspend each target in 30 µl of 3X SSC, 0.1% SDS (these may be hard to resuspend, place in 37¡C heat block and vortex if necessary. This may take 30-45 min.).
Mix both Cy5 and Cy3 resuspended target, add 4 µl of tRNA (8 mg/ml) and mix well by vortexing.
Boil for 5 minutes in a heat block.
Incubate for 5 minutes at 50¡C.
Spin briefly.

Step 7b - Hybridization Pipette 50 µl of probe onto slide and drop cover slip (use the big one so that it will cover the entire array) onto the liquid. Try to avoid bubbles as they exclude the hybridization solution.
Add water to the holes in the hybridization chamber.
Assemble the chambers and submerge right side up in a 50¡C water bath, allow hybridizing for 20-24 hours.

Step 7c - Wash Disassemble hybridization chambers with the right side up.
Remove coverslip and immediately place slide in 0.1X SSC, 0.1% SDS at room temperature for 8 minutes with agitation.
Transfer to 0.1X SSC for 5 minutes with agitation.
Note: Tranfer slide by slide (do not transfer the whole rack).
Rotate slides 180¡ along the long edge when transfering.
Repeat 0.1X SSC wash 2 more times.
Dry by placing slides in a rack and spinning in a centrifuge for 2 min @ 1 krpm and scan immediately or store in the dark until scanning.
specified_biomaterial_action
Preparation of Magnetic Beads

* Prepare the day before use *

1. Take 50 ul of beads (4 x108 beads/ml stock e.g. 2X 107 beads per sample) and place in a 15 ml Falcon tube. Use Dynabeads M-450 pre-coated with rat anti-mouse IgG-2a; Cat.#110.13.
2. Spin for 1 minute at speed 6 (~3000 rpm) in a tabletop centrifuge (Sorvall RT6000).
3. Remove supernatant with a pipette and resuspend in 10 ml PBS containing 5mg/ml BSA (make immediately before use from Sigma BSA powder, cat. A-3350).
4. Wash again.
5. Incubate overnight with antibody on a rotating platform at 4¡C (Use 1 ul of anti-Myc 9E11 antibody plus 250 ul PBS + 5 mg/ml BSA per 50 ul of beads).
* Note: The 9E11 antibody we are using has been purified from acites and concentrated. The amount used has been determined empirically so that the beads are saturated.
6. Spin for 1 minute at speed 6 (~3000 rpm) in a tabletop centrifuge (Sorvall RT6000).
7. Remove supernatant with a pipette and resuspend in 10 ml PBS containing 5mg/ml BSA (make immediately before use, as above).
8. Wash again.
9. Resuspend each sample in 30 ul PBS containing 5mg/ml BSA.

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Step 2b - Immunoprecipitation

1. Set up a new tube on ice containing: 500 ul of yeast whole cell extract (yWCE) and 30 ul of a suspension of washed magnetic beads pre-bound to anti-Myc antibody (see Appendix 1).
* Vortex the beads well before removing each 30 ul aliquot to ensure equal amounts of beads are added to each tube and that the beads remain in suspension. Set aside 5 ul of yWCE in a separate tube (to label as a control later) and store it and the rest of the yWCE at -20¡C.
2. Incubate overnight on a rotating platform at 4¡C.

Step 3 - Bead Washing

* Work in the Cold Room *

1. Wash beads using appropriate device (e.g. MPC-E magnet, Dynal), as follows:
* Put the first 6 tubes into magnet, invert the tubes once, open the tubes and aspirate the supernatant using a vacuum (also aspirate what is left in the cap), add the appropriate washing solution, close the tubes and put them back on the rotating platform. Proceed with the next 6 tubes and so on. Don't forget to turn the rotator on while you are aspirating the supernatant from the next set of tubes etc.
* For this step, you don't need to add protease inhibitors to the lysis buffer.
2. Wash 2 times with 1 ml lysis buffer.
3. Wash 2 times with 1 ml lysis buffer containing an additional 360 mM NaCl
* 720 ul of 5 M NaCl in 10 ml lysis buffer - the final concentration of NaCl is 500 mM.
4. Wash 2 times with 1ml wash buffer.
5. Wash once with 1 ml TE.
6. After you have removed the TE by aspiration, spin the tubes for 3 minutes at 3000 rpm and remove any remaining liquid with a pipette.

Step 3a - Elution from beads and reversal of cross links

7. Add 50 ul elution buffer, vortex briefly to resuspend the beads and incubate at 65¡C for 10 minutes. Vortex briefly every 2 minutes during the incubation.

*The next steps should be done at room temperature*

8. Spin for 30 seconds at maximum speed and transfer 30 ul of supernatant to a new tube. Discard the rest (unless have a special reason to keep it).
9. Add 120 ul of TE/SDS to the supernatant in the new tube in order to reverse the crosslinking reaction.
10. Also add 95 ul of TE/SDS to 5 ul of yWCE (prepare one yWCE for each IP).
11. Incubate overnight at 65¡C in an incubator.
grow
 
The 6361 intergenic regions were amplified using the Yeast Intergenic Region Primers (Research Genetics). 50µL PCR reactions were performed in 96-well plates with each primer pair with the following conditions: 0.25 µM of each primer, 20 ng of yeast genomic DNA, 250 µM of each dNTP, 2 mM MgCl2, 1X PCR buffer (Perkin Elmer), and 0.875 units of Taq DNA polymerase (Perkin Elmer). PCR amplification was performed in MJ Research Thermocyclers beginning with 2 minute denaturation at 95°C, followed by 36 cycles of 30 seconds at 92°C, 45 seconds at 52°C, and 2 minutes at 72°C, with a final extension cycle of 7 minutes at 72°C. 1 µL of each PCR reaction mix was then reamplified in a 100 µL PCR reaction using universal primers (Life Technologies) with the same reagent concentrations and the following thermocycling conditions: 3 minutes at 94°C, followed by 25 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 1 minute at 72°C, with a final extension cycle of 7 minutes at 72°C. Each PCR product was verified by gel electrophoresis. The PCR products were then isopropanol precipitated, washed with 70% ethanol, dried overnight, and resuspended in 20 µL of 3X SSC. The resuspended DNA was transfered to 384 well plates and printed on GAPS-coated slides (Corning) using a Cartesian robot (Cartesian Technologies). The printed slides were rehydrated, snap-dried, and UV crosslinked in UV Stratalinker (Stratagene) set at 60 mJoules. The slides were then stored under vacuum for at least 2 days prior to hybridization. Quality Controls were performed as described in: http://jura.wi.mit.edu/young_public/regulatory_network/QC%20of%20arrays.pdf