Schott Nexterion Epoxy troubleshooting

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Schott Nexterion Epoxy troubleshooting

Postby aferreira » Mar 12 2009 8:17 pm

I purchased spotted arrays from Stanford. As suggested in the Schott protocols I was sent I baked the arrays 120 degrees 30 minutes (oligo arrays) and then immobilized in a humidity chamber 30 minutes.
I followed the washing and ethanolamine block suggested in the manufacturers protocols as well.

I labelled quality checked RNA with invitrogen protocols and measured the incorporation efficiency on a nanodrop which was as it should be.

I hybridized in 35% formamide, 5X SSC, 0.5% SDS, 2.5X Denhardts for 18 hours at 42 degrees and washed as follows:

2 x 5 min 2XSSC, 0.1%SDS
2 X 5 min 0.5XSSC, 0.1% SDS
2 x 2.5 min 0.1XSSC

Results: I had very few hybridized spots although I had a somewhat clean background. What worried me was that the spots were black in the middle (of varying sizes) with a light halo around them (just slightly brighter than the backgound)... some looked like very large black holes.

My first guess would be that the hybridization or wash was too stringent. Anyone have any other suggestions?

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