Bauer Core Standard Protocol
Title: Preparation of GeneChips and Target Hybridization Cocktail
Pages: 4
Revision:
Date: August 26, 2003
Author(s): Jennifer A. Couget
Reviewers: Shufen Meng
Contact: affyinfo@cgr.harvard.edu
Comment:
1. Purpose
This protocol describes the preparation of Affymetrix GeneChips and Target Hybridization
Cocktail that may be used for a Test array and follow-up GeneChips of interest.
2. Materials
Affymetrix GeneChips
Labeled cRNA
1X Hybridization Buffer
2X Hybridization Buffer
20X Eukaryotic Hybridization Control Kit includes Oligo B2 (Affymetrix, p/n 900299)
Oligo B2 (extra vial may be purchased if desired Affymetrix, p/n 900301)
Herring Sperm DNA 10 mg/ml (Promega, p/n D1811)
Acetylated BSA 50 mg/ml (Invitrogen, p/n 15561-020)
DEPC-water (Ambion, p/n 9920)
1.7 ml RNase-free tubes (Sorenson, p/n 16070)
GeneMateTube-tabs (ISC Bioexpress, p/n C-3271-2)
0.1-10ul sterile, filter-free pipette tips (sharp tip/not beveled) (Marsh, p/n TN10RS)
Lab Tape or Diversified Biotech Tough Spots (ISC, p/n L-1000-6)
3. Instrumentation
Heat blocks with stable temperatures at 45C, 65C, and 99C.
Benchtop mini-fuge
Microcentrifuge
4. Reagent preparation
2X Hybridization Buffer (50ml)
12X MES Buffer
5M NaCl (Ambion, p/n 9760G)
0.5M EDTA (Ambion, p/n 9260G)
10% Tween-20 (Pierce, p/n 28320)
DEPC-water (Ambion , p/n 9920)
8.3 ml
17.7 ml
4 ml
0.1 ml
19.9 ml
Store at 4C. Discard if yellow.
1X Hybridization Buffer
Mix one part 2X Hybridization Buffer with one part DEPC-water.
Store at 4C. Discard if yellow.
5. Procedure
5.1
It is usually best to make all cocktails for a set of arrays fresh to minimize freeze-thawing
effects on cRNA and at the same time to minimize day-to-day variability.
5.2
The final concentration of the cRNA in the hybridization cocktail must be no less than
0.05 µg/µl. If you followed the recommended fragmentation reaction (20 µg of cRNA
fragmented in 40 µl), you meet these requirements.
Equilibration of Reagents
5.3
Equilibrate arrays to room temperature approximately 15 to 20 minutes before use.
Failure to do so may cause array septa to crack and damage chip irreversibly.
5.4
Pre-heat oven to 45C (takes about 5 minutes).
5.5
Place 2X and 1X Hybridization Buffers at room temperature for 5 to 10 minutes before
using.
5.6
Heat 20X Eukaryotic Hybridization Controls and OligoB2 to 65C in standard 1.7 ml
eppendorf tubes for 5 to 10 minutes to ensure proper mixing. Quick-vortex to mix and
quick-spin in mini-fuge.
5.7
Heat Herring Sperm DNA and BSA at 37C for 5 to 10 minutes. Quick-vortex and quickspin in mini-fuge.
5.8
Keep fragmented cRNA on ice until ready to use. If cRNA is frozen, thaw at 37C for 5 to
10 minutes, quick-vortex, quick-spin in mini-fuge, and place on ice.
Preparing the GeneChip
5.9
Remove GeneChip from packaging and inspect it for manufacturing defects (i.e.,
scratches on glass or wafer).
5.10
Inspect GeneChip so that you may easily read the GeneChip type, lot#, and expiration
date. Flip GeneChip directly over. The narrow portion of the chip is positioned at the top.
The septa are positioned at upper right and lower left. You must use 2 pipette tips, one
for venting and one for filling, when loading a GeneChip.
5.11
Insert sharp, filter-free 0.1-10 µl tip into septum at upper right to vent.
5.12
Hold GeneChip upright and fill GeneChip with 1X Hybridization Buffer through septum
at lower left:
Format
Mini/Micro
Midi Format
Standard Format
Example
Test3 Chip
Human Focus Chip
Genome Chip
Fill Volume (µl)
100
200
300
5.13
Insert GeneChips into GeneChip cartridge carrier. Prepare balance carrier with
cartridges.
5.14
Balance and insert carriers into pre-heated 45C oven. Make sure carriers are properly
secured over notches and will not fall out.
5.15
Close door to oven and make a full 360C turn with handle to close door securely.
5.16
Set rotation speed to 60 rpm.
5.17
Pre-hybridize arrays for a minimum of 10 minutes and no longer than 25 minutes. Do not
allow arrays to dry.
Preparation of Target Hybridization Cocktail
5.18
Mix all components of the target hybridization cocktail except cRNA. If an error is made,
you will not have to re-do the fragmentation step.
5.19
Add the following to a 1.7-ml eppendorf tube, quick-vortex to mix, and quick-spin in
mini-fuge.
Component
Volume (µl)
DEPC-water
85
2X Hybridization Buffer
150
3nM Oligo B2
5
20X Eukaryotic Hybridization Controls
15
10 mg/ml Herring Sperm DNA
3
50 mg/ml Acetylated BSA
3
5.19.1 Add last: Fragmented cRNA (20 µg)
Final volume
39
300
Final Concentration
1X
50 pM
1.5, 5, 25,100 pM mix
0.1 mg/ml
0.5 mg/ml
0.06 µg/µl
5.19.2 Visually check that final volume in tube is 300 µl.
5.20
Slide tube-tabs onto 1.7-ml tubes to prevent tubes from opening under pressure.
5.21
Incubate hybridization cocktails at 99C for 5 minutes. Carefully remove cocktails from
heat block; remove tube-tabs that may be under pressure, and quick-spin tubes in minifuge to bring down any concentration under lids.
5.22
Incubate hybridization cocktails at 45C for 5 minutes to equilibrate to array
temperature.
5.23
Place hybridization cocktails in properly balanced microcentrifuge. Align tubes so that
debris will form at the same spot in each tube.
5.24
Spin hybridization cocktails at maximum speed for 5 minutes at room temperature to
remove insoluble debris.
5.25
Carefully remove hybridization cocktails from microcentrifuge. Place in rack at room
temperature. Do not disrupt any debris at the bottom of the tube. If you disrupt the
debris, go back to step 5.25.
5.26
Remove GeneChips from oven and inspect GeneChips for manufacturing defects (i.e.,
leaks or cracks evidenced by dried salt crystals on glass or septum).
5.27
Remove 1X Hybridization Buffer from GeneChip, using the same technique described in
steps 5.10 to 5.12. Remove all buffer solution from chamber by drawing up fluid by
pipette and disposing. Repeat a second time to remove residual buffer and air. Replace
vent tip and pipette tip if necessary.
5.28
Immediately fill chamber with appropriate sample hybridization cocktail volume for
GeneChip. Avoid insoluble debris at the bottom of the tube.
Format
Mini/Micro
Midi Format
Standard Format
Example
Test3 Chip
Human Focus Chip
Genome Chip
Sample Volume (µl)
80
130
200
5.29
Upon filling, a small single meniscus should be visible through the glass window, with
few to no bubbles. Mini/Micro formatted arrays are too small to visualize this.
5.30
Remove vent, and check GeneChips for leaking through glass and septa.
5.31
Seal both septa with lab tape or Tough-Spots.
5.32
Repeat steps 5.13 to 5.16.
5.33
Hybridize arrays for 16 hours. Do not allow arrays to over-hybridize or dry.
5.34
Place remaining cocktail at –20C for overnight storage.
5.35
After hybridization, recover cocktail from GeneChip and place in original tube. Store at 20C for short-term storage or -80C for long-term storage.