Eukaryotic

Eukaryotic Target Hybridization

Reagents and Materials Required

The following reagents and materials are recommendations

• DEPC- Treated Water. Ambion Cat.#9922
• Bovine Serum Albumin (BSA) solution (50 mg/mL), Invitrogen Life Technologies, P/N
• 15561-020
• Herring Sperm DNA, Promega Corporation, P/N D1811
• GeneChip Eukaryotic Hybridization Control Kit, Affymetrix, P/N 900454 (30 reactions) or
• P/N 900457 (150 reactions), contains Control cRNA and Control Oligo B2
• Control Oligo B2, 3 nM, Affymetrix, P/N 900301 (can be ordered separately)
• 5M NaCl, RNase-free, DNase-free, Ambion, P/N 9760G
• MES hydrate SigmaUltra, Sigma-Aldrich, P/N M5287
• MES Sodium Salt, Sigma-Aldrich, P/N M5057
• EDTA Disodium Salt, 0.5M solution (100 mL), Sigma-Aldrich, P/N E7889
• DMSO, Sigma-Aldrich, P/N D5879
• Surfact-Amps 20 (Tween-20), 10%, Pierce Chemical, P/N 28320

Miscellaneous Supplies

• Hybridization Oven 640, Affymetrix, P/N 800138 (110V) or 800139 (220V)
• Sterile, RNase-free, microcentrifuge vials, 1.5 mL, USA Scientific, P/N 1415-2600 (or equivalent)
• Micropipettors, (P-2, P-20, P-200, P-1000), Rainin Pipetman (or equivalent)
• Sterile-barrier pipette tips and non-barrier pipette tips
• Heatblock (Fisher-scientific)
• Thermometer

Reagent Preparation

12X MES Stock Buffer

(1.22M MES, 0.89M [Na+])

For 1,000 mL:

64.61g of MES hydrate

193.3g of MES Sodium Salt

800 mL of Molecular Biology Grade water

Mix and adjust volume to 1,000 mL.

The pH should be between 6.5 and 6.7. Filter through a 0.2 μm filter.

2X Hybridization Buffer

(Final 1X concentration is 100 mM MES, 1M [Na+], 20 mM EDTA, 0.01% Tween-20)

For 50 mL:

8.3 mL of 12X MES Stock Buffer

17.7 mL of 5M NaCl

4.0 mL of 0.5M EDTA

0.1 mL of 10% Tween-20

19.9 mL of water

Store at 4 °C, and shield from light.

Eukaryotic Target fragmentation and Hybridization

1. Calculate the necessary amount of cRNA 20ug (cRNA concentration at least 0.625ug/ul ) Place in 0.5ml tubes. Adjust volume to 32ul with nuclease free water.

2.Add 8ul of 5x Fragmentation buffer to each sample. Vortex, spin briefly.

3. Incubate at 94°C for 35 minutes in the heat block.

4.While cRNA is fragmenting, take out Gene Chips and place them at room temperature.

It is important to allow the chips to equilibrate to room temperature completely.

Specifically, if the rubber septa are not equilibrated to room temperature, they may

be prone to cracking, which can lead to leaks. Usually takes about 20 minutes.

5. . Incubate the probe array filled with 1X Hybridization Buffer at 45°C for 10 minutes

with rotation.

49 Format (Standard) 200ul

64 Format 200 ul

100 Format (Midi) 130 ul

169 Format (Mini) 80 ul

400 Format (Micro) 80 ul

Place Chips at 45°C in Hybridization oven, rotating at 60 rpm. for 10 minutes.

6. When 35 min is up remove samples from heat block and spin down .

7.Add the following reagents

5ul of Oligo B2

15 ul of 20x Eukaryotic Controls (It is imperative that frozen stocks of 20X GeneChip Eukaryotic Hybridization Controls are heated to 65°C for 5 minutes to completely resuspend the cRNA before

aliquotting)

3ul of herring Sperm DNA

3ul of BSA

84 ul of Nuclease free water

150 ul of 2x Hybridization Buffer

Vortex,Spin briefly..

8.. Heat the hybridization cocktail to 99°C for 5 minutes in a heat block.

9. Transfer the hybridization cocktail that has been heated at 99°C, to a 45°C

oven for 5 minutes.

10. Spin hybridization cocktail(s) at maximum speed in a microcentrifuge for 5 minutes to

remove any insoluble material from the hybridization mixture.

11.Remove the buffer solution from the probe array cartridge and fill with appropriate

volume (Table 2.2.2) of the clarified hybridization cocktail, avoiding any insoluble

matter at the bottom of the tube.

12.Place probe array into the Hybridization Oven, set to 45°C.

Avoid stress to the motor; load probe arrays in a balanced configuration around the

axis. Rotate at 60 rpm.

13.Hybridize for 16-18 hours.