GenomeBC Forestry, Arabidopsis oligo array hybridization protocol, March 2005, NM
Arabidopsis oligo array hybridization
Day 1
Set two waterbaths or two heatblocks to 42ºC and 65ºC or, alternatively…
Programme PCR machine
Reverse-Transcriptase Reaction (direct labeling)
Add: 8.0 µl
1.5 µl
3.0 µl
1.0 µl
4.0 µl
0.3 µl
18.2 µl
5X First Strand Buffer
T17 (Anchor) primer (100 µM)
dNTPs (without dTTP) (6.7 mM each)
dTTP (2 mM)
DTT (0.1 M)
human spike-RNA
x µl RNA (60/80 µg in less than 18.2 µl)
y µl H2O (RNAse free) to reach a final volume of 37 µl
Protect tubes from light…
Add: 1.0 µl Cy-dUTP (1 nM)
Incubate: 65ºC, 5 min
42ºC, 5 min
Add: 1 µl
2 µl
RNAse Inhibitor (40 U/µl)
SuperScript II RT (200 U/µl)
Final reaction volume: 40 µl
Incubate: 42ºC, 2 - 3 h
Add: 8 µl
NaOH (1M)
Incubate: 65ºC, 15 min
Add: 8 µl HCl (1M)
4 µl Tris-HCl, pH7.5 (1M)
40 µl H2O
Use 44 µl from a
1:10 Tris pH 7.5:
H2O sol’n
Final volume: 100 µl
Probe Purification
Purify using the Qiaquick PCR purification kit according to the manufacturers protocol
Briefly…
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Add 5 volumes PB Buffer to sample (500 µl)
Apply sample to column and centrifuge 30-60 s
Discard flow-through
Add 750 µl PE Buffer and centrifuge 30-60 s
Discard flow through
GenomeBC Forestry, Arabidopsis oligo array hybridization protocol, March 2005, NM
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Centrifuge again 30-60 s
Place column into a clean µctube
Add 50 µl EB Buffer and centrifuge 60 s
Repeat into same tube
Combine samples to be hybridized on the same array
Add 1 µl Cy5-labeled gfp (corner) marker
Final volume: 201 µl
Ethanol precipitation
Add: 0.1 x volume (20 µl) sodium acetate (3M)
2.5 x volume (500 µl) ethanol (100 %)
Incubate: -20ºC, overnight
Day 2
Set shaking water bath to 48ºC, 40 rpm
Set heating block to 65ºC
Set heating block to 95ºC
Set Eppendorf (refrigerated desktop) centrifuge to 4ºC
Pre-warm hybridization buffer (Ambion #1) to 65ºC
Array Pre-Hybridization
Prepare prehybridization solution (70 ml per Joplin jar, 2 slides per Joplin jar)
5 X SSC
0.1 % SDS
0.2 % BSA
25 ml 20 X SSC
1 ml 10 % SDS
0.2 g BSA (Sigma)
ad 100ml with H2O (74 ml)
Pre-warm prehybridization solution to 48ºC
Incubate arrays in Joplin jars in prehybridization solution for 45 - 60 min in shaking
water bath at 48ºC (Figure 1)
Probe preparation (continued from Ethanol Precipitation)
Spin precipitated probe: 14,000 rpm, 4ºC, 20 min, Eppendorf centrifuge
Remove supernatant completely (use vacuum)
Wash pellet with 200 µl ethanol (70 %)
Spin: 14,000 rpm, 4ºC, 15 min, Eppendorf centrifuge
Remove supernatant completely (use vacuum)
GenomeBC Forestry, Arabidopsis oligo array hybridization protocol, March 2005, NM
Air-dry pellet shortly (about 1 min), do not over-dry
Resuspend pellet in 3.5 µl EDTA (10 mM)
Denature at 95ºC, 1-2 min
Add 50 µl pre-warmed hybridization buffer (Ambion #1)
Leave at 65ºC until used
Array Preparation (continued from Array Pre-Hybridization)
Discard prehybridization solution
Wash slides twice in H2O for 10 sec. at RT
Dip slides 5 times in isopropanol (100%)
Spin slides immediately: 2000 rpm, 3 min, RT, Haereus centrifuge (in 50 ml Falcon tube
with a Kimwipe paper stuffed at the bottom)
Transfer slides to a dry jar and keep until used
Array Hybridization
Re-set shaking water bath to 42ºC, 40 rpm
Add 20 µl H20 to wells in the hybridization chamber (Figure 2)
Place slide in hybridization chamber
Add denatured probe to the slide (Figure 3)
Put on cover slip (untreated glass cover slips) (Figure 4)
Seal hybridization chamber
Submerge in water bath
Incubate: 42ºC, 40 rpm (along the long axis of hybridization chamber), overnight (~14h)
Day 3
Washing Hybridized slides
Prepare Wash Solutions:
Array-Wash 1:
2 X SSC
0.5 % SDS
20 ml 20 X SSC
10 ml 10% SDS
ad 200 ml with H2O
Array-Wash 2:
0.5 X SSC
0.5 % SDS
5 ml 20 X SSC
10 ml 10 % SDS
ad 200 ml with H20
Pre-warm Wash Solutions to 42 ºC
Disassemble hybridization chamber
GenomeBC Forestry, Arabidopsis oligo array hybridization protocol, March 2005, NM
Remove cover slip by gently dipping the slide in Array-Wash 1 in Joplin jar
Wash slides in Array-Wash 1 for 15 min, 42ºC, shaking water bath, in Joplin jars
Wash slides twice in Array-Wash 2 for 15 min, 42ºC, shaking water bath, in Joplin jars
Wash slides in 0.1X SSC for 1 min at RT with shaking
Spin slides: 2000 rpm, 3 min, RT, Haereus centrifuge (in 50 ml Falcon tube with a
Kimwipe paper stuffed at the bottom)
Store slides in the dark until scanned (Figure 5)
Some Recipes:
(Each Joplin jar holds ~70 ml)
2X SSC (200ml)
20 ml 20X SSC
180 ml dd H2O
0.1X SSC (200 ml)
10 ml 2X SSC
190 ml dd H2O
0.1X SSC (200 ml)
1 ml 20X SSC
199 ml dd H2O
20X SSC
175.3 g NaCl
88.2 g Na-citrate
ad 1L H2O
pH 7.0 with HCl
GenomeBC Forestry, Arabidopsis oligo array hybridization protocol, March 2005, NM
GenomeBC Forestry, Arabidopsis oligo array hybridization protocol, March 2005, NM