Maitreya Dunham and Matt
Brauer June 2005
From various Elizabeth
Winzeler papers, the Affy manual, and the Enzo manual.
Use high-quality DNA.
Bring 10 ug DNA to 15.8 ml total volume in 10 mM Tris pH 8.
Add:
2 ml |
10X one-phor-all buffer |
1.2 ml |
25 mM CoCl2 |
Quickly add:
1 ml |
0.15 U diluted DNase (1 ml DNase+5.7 ml 1X one-phor-all) |
If doing several
reactions, you may want to do them in batches.
Incubate 37C exactly 5 min.
Incubate 100C 15 min.
Run 1 ml on a 2% gel for 50 min at 50 V. Also run a similar amount of undigested
DNA to check quality.
The DNA should be all in a
smear ~50 bp.
If it is not, start again.
For labeling, use the
BioArray Terminal Labeling Kit (Enzo 42630)
Add, at room temperature:
20 ml |
5X reaction buffer |
10 ml |
10X CoCl2 |
1 ml |
Biotin-ddUTP |
2 ml |
Terminal Deoxynucleotide
Transferase |
48 ml |
water to 100 ml |
Incubate 37C 15 minutes to
1 hour.
Ice.
Stop with 5 ml 0.2 M EDTA.
Add:
27 ml |
water |
150 ml |
2X hybe buffer |
15 ml |
10 mg/ml BSA |
3 ml |
10 mg/ml salmon sperm
DNA |
5 ml |
3 nM control oligo B2 |
Incubate 99C 5 min.
While incubating, load
array (see loading instructions below) with 200 ml 1X hybe buffer (see recipes) and prehybe in the
oven for 10 min at 45C.
Incubate probe 45C 5 min.
Spin 5 min.
Remove the prehybe buffer
from the array and load with 200 ml probe, avoiding any debris at the bottom of the tube.
Tape over septa to prevent
leakage.
Put arrays in oven at 45C,
60 RPM.
Note time. Hybe 20 hours.
Put a 200 ml filter tip in one of the septa as an air
release. Pipet up your solution
with a 200 ml filter tip. Holding the array so the air release is
upward, puncture the other septum and slowly pipet to fill. Withdraw the tip. You should have a bubble in the array
chamber. Remove the air release
tip. It should not have any
solution in it.
Place the array in the
plastic holder tray. Snap it into
the holders on the oven. Balance
with another tray and trash arrays.
Make all wash buffers
before starting.
For each wash station,
make about 0.5 L Wash A and Wash B.
For each array, make 1.2
ml (in two 600 ml aliquots) SAPE and
600 ml Antibody solution.
Log in to the Affy
computer as maitreya with password affy123.
Follow directions for
starting and priming the fluidics, starting on page 2.3.7 of the manual. (Make sure you use the instructions for
the correct model of wash stations.
A few little things change.)
Follow the directions for
the washes, using the EukGE-WS2v4 protocol.
When done, remove the
arrays from the fluidics station.
Dab some whiteout on the septa to seal them. Store slides in the dark until they are scanned.
Scan.
Burn the data to CD.
Wash and shut down
fluidics station.
2X Hybe buffer
50 ml
8.3 ml |
12X MES (to 200 mM) |
17.7 ml |
5 M NaCl (to 2M Na+) |
4 ml |
0.5 EDTA (to 40 mM) |
100 ml |
10% Tween-20 (to 0.02%) |
water to 50 ml
Store 4C in dark.
Wash buffer A
1 L
300 ml |
20X SSPE (to 6X, i.e.
0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA) |
1 ml |
10% Tween-20 (to 0.01%) |
water to 1 L
Filter sterilize.
Wash buffer B
1 L
83.3 ml |
12X MES (to 100 mM) |
5.2 ml |
5 M NaCl (to 0.1 M Na+) |
1 ml |
10% Tween-20 (to 0.01%) |
water to 1 L
Filter sterilize.
Store 4C in dark.
2X Stain Buffer
250 ml
41.7 ml |
12X MES (to 200 mM) |
92.5 ml |
5 M NaCl (to 2 M Na+) |
2.5 ml |
10% Tween-20 (to 0.1%) |
water to 250 ml
Filter sterilize.
Store 4C in dark.
12X MES
100 ml
6.461 g |
MES hydrate (to 1.22 M
MES) |
19.33 g |
MES sodium salt (to 0.89
M Na+) |
water to 100 ml
Filter sterilize.
pH should be between 6.5
and 6.7.
Store 4C in dark. Discard if it turns yellow.
20X SSPE
3 M |
NaCl |
0.2 M |
NaH2PO4 |
20 mM |
EDTA |
SAPE (aka stain 1 and 3)
1.2 ml
12 ml |
1 mg/ml R-streptavidin
phycoerythrin (to 10 ug/ml) |
240 ml |
10 mg/ml BSA (to 2
mg/ml) |
600 ml |
2X stain buffer (to 1X) |
water to 1.2 ml
Make the day of use. Store 600 ml aliquots at 4C in dark until use.
Antibody solution (aka
stain 2)
600 ml
6 ml |
10 mg/ml goat IgG (to
0.1 mg/ml) |
120 ml |
10 mg/ml BSA (to 2
mg/ml) |
3.6 ml |
0.5 mg/ml
anti-streptavidin antibody (goat), biotinylated (to 3 ug/ml) |
300 ml |
2Xstain buffer (to 1X) |
water to 600 ml
Make the day of use. Store 600 ml aliquots at 4C in dark until use.