P-FPMI-45 - P-FPMI-45

Type
hybridization
Description
Hybridization using Jack Bell 21K Human Microarray slides


a. Pre-hybridization and pretreatment of slides:


i. Prepare 500 ml pre-Hybridization Solution in MilliQ water as follows:


125 ml 20 x SSC (final conc 5X SSC)

5ml 10% SDS (final conc 0.1% SDS)


1g BSA (solid) (final conc 0.2% BSA)


ii. Warm the pre-hyb solution to 37C to dissolve.

iii. NOTE: Use a cleaned pair of tweezers EVERY TIME you touch the slides. Pre-hybridization buffer contains BSA and will leave a large streak down the slide, and ruin the subsequent scans.

iv. Place slides into a clean wash vessel, pour approximately 450 ml of pre-hyb buffer in and incubate at 48 C for 45 mins. Make sure that the solution covers the slides.

v. Wash the slides twice by dipping up and down around 10 times each in MilliQ water in clean wash vessels.

vi. Wash the slides once by dipping up and down around 10 times in RT isopropanol.

vii. Spin dry the slides at 1,000 rpm for 5 mins at RT.

viii. NOTE: Make sure the slides are used within one hour of spinning dry. Atmospheric humidity will cause problems.


b. 1 mm LifterSlip cleaning (Eerie Scientific: 25X60I-2-4789)


i. Dip lifter slip in clean dH2O

ii. Dip LifterSlip in 100% EtOH

iii. Blow dry with clean air. You may use a Kimwipe to remove any other blotches you are unable to remove by the above method


Hybridization


i. After analyzing your labeled samples by absorbance, take 20 pmol of Cy3-labelled sample and 20 pmol of Cy5-labelled sample and combine the samples to be hybridized on one slide together.

ii. Add 1 µl GFP labeled probe.

iii. Speed vac the samples, DO NOT OVERDRY, try to leave around 1-2 l in the tube (dried nucleic acids dissolve poorly in hybridization buffer). In case the sample goes completely dry add 1 – 2 l NF- water to the pellet and tap and re-suspend.

iv. Make up hybridization solution master mix for all the slides.

v. For each 80 l hybridization solution add the following in the master mix:


72.8 l DIG Easy hyb solution

3.6 l salmon sperm DNA (10 mg/ml)[Ambion]

3.6 l tRNA (10 mg/ml) [Ambion]

0.28 l poly dA (1 g/ml) [Ambion]

Dry down 8g of human cot-1 DNA [Invitrogen, catalog # 15279-011] and resuspend in the above mix


[If you want more volume of hyb buffer / slide depending on slide, keep the ratios of the components consistent]


vi. Add 80 l of the hybridization buffer to each tube with the speed vac-concentrated samples.

vii. Mix the sample with the hybridization buffer and denature the sample at 90C for 1 min.

viii. Keep the sample on ice for at least 1 min.

ix. Remove it from ice, spin down quickly using table top microcentrifuge and keep samples covered from light at RT or warmer until ready to put on slide.

x. Place the pretreated cover slips on slides, one at a time

xi. Pipet all of the sample onto the spotted side of the pretreated slide under the short side of the lifter slip. Wait until the sample reaches the far side of the lifterslip and if required inject the last 5 -10 l of the sample under the far side of the lifterslip. [NOTE: Keep in mind the spotted side of the slide with respect to the barcode]

xii. Place the slide in a corning cassette with 10 l NF-H2O in each well of the cassette and close.

xiii. Seal Hybridization Chamber. Incubate on a level surface in a humidified chamber at 37°C for at least overnight (18 hrs).




Washing


i. Immerse the pair of slides in 1 X SSC and slide the arrays past one another. NOTE: wash forceps in 70% EtOH between samples and between the various steps to reduce streaking.

ii. Pre-warm 1 X SSC / 0.1 % SDS at 37 C, wash the slides twice for 5 mins each at 37°C in pre-warmed 1X SSC / 0.1% SDS with gentle occasional agitation ( make one litre of 1XSSC/0.1% SDS and incubate at 37°C to pre-warm it overnight)

iii.

1 L of 1X SSC/ 0.1% SDS


20 X SSC 50ml

10% SDS 10ml

MQ Water 940 ml


iv. Wash the slides in 1X SSC for 3 mins followed by wash in 0.1X SSC for 3 mins to completely remove the SDS.


1000 ml of 1X SSC 500 ml of 0.1X SSC


20X SSC 50ml 20X SSC 2.5ml

MQ Water 950 ml MQ Water 997.5ml



v. Place the slides in a 50 ml falcon tube, DO NOT PLACE LID ON THE TUBE. Keep some kimwipe on the bottom of the tube, place barcode facing the bottom of the tube and place in centrifuge with barcode facing out.

vi. Spin dry the slides at 1,000 rpm for 5 min in room temp in the 50 ml tube.

vii. Store the slides in dark before scanning and analysis.

Parameters
Chamber type, Quantity of label target used, Temperature, Time, Volume
Links
All experiments using protocol P-FPMI-45: (E-FPMI-11, E-FPMI-13, E-FPMI-4, E-FPMI-5, E-FPMI-6, E-FPMI-7)