6 protocols
AccessionType
feature_extraction
ll the slides for a same project are scanned at a constant PMT power (i.e. 650V for 635nm and 620V for 532nm).
hybridization
Slide preparation In a Coplin jar, prepare 50 ml of pre-hybridization solution :
SSC 20 X 12,5 ml
BSA 10 % 5 ml
SDS 10 % 0,5 ml
H2O 32 ml
Pre-heat this solution at 42 °C and préhybridize slides at 42 °C for at least 1 hour.
Wash the slides in milli-Q water, and then isopropanol.
Blow-dry the slides.
THE SLIDES MUST BE USED WITHIN 1 HOUR!

Hybridization
Prepare 2X hybridation mix (35 µl per slide) :
SSC 20x 50 µl
SDS 10% 2 µl
Formamide 50 µl

Denature the probe 1 mn at 95°C and keep on ice.
Add one volume of 2X hybridation mix, mix and centrifuge.
Put the slide in the Corning hybridization chamber, and place a cover slip on it.
Pipet the probe between the slide and the cover slip.
Put 10 µl of water in the two holes and close the chamber.
Incubate in a water bath at 42°C over-night (16h)

Washes
Soak in 2X SSC, 0,1% SDS at 42°C to remove the cover slip, then wash in :
- 2X SSC, 0,1% SDS (42°C) 4 min
- 1X SSC 4 min
- 0,2X SSC 4 min
- 0,05X SSC 1-4 min
Blow dry the slide or centrifuge for 2 min at 2000 rpm.
Store in a dry and dark place until scanning.
labeling
5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample: 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy3-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
labeling
5 µg of aRNA (8µl) mixed with 2 µl of random nonamers 1µg/µl and 0.5 µl of RNAse Out 40 U/µl, denatured 10 min at 70 °C and chilled on ice. The followig components were added to the sample: 4 µl of first strand buffer 5 X, 1 µl of 10 mM dNTP/2mM dCTP, 2 µl of 0.1 M DTT, 1.5 µl of Cy5-dCTP (Amersham Pharmacia Biotech, 25nmol tube, PA5502), 1 µl of SuperScript II RT 200U/µl. Then it was incubated at 42°C for 2.5 hours and chilled on ice. The sample was denaturated by adding 2 µl of NaOH 2.5M and incubated at 37 °C for exactly 15 min, then was added 10 µl of 2M MOPS and put on ice. The dyes were purified with the QIAquick PCR Purification Kit (QIAGEN).
nucleic_acid_extraction
grow
Media : Arabidopsis medium Somerville&Estelle 1990 (0% sucrose)
hygrometry : 70%
Temperature : 20°C
Light : 4h Flash but none during growth (prelevment under green light)