Greetings! I run microarray at many condition, so I want to know many things.

1. Why we have to hybridization at 42 degree?

I ran experiment, hybridization durong O/N(16~20h), it was good fluorescence that overnight hybridization at RT better than 42 degree overnight.

I heard about 42 degree is good condition to hybridization buffer that include formamide, so we have to run at 42 degree by just that reason?

2. why we have to bake at 80 degree, dry oven?

actually, 80 degree, overnight woth water(water drought durong baking) decreases coffee ring effects. parafilm is weak at 80 degree(heat) but spot was good than 80min dry oven.

and, when I baked 60 degree oven(with water), it was baked but slide saw one of unspecific dot. (left-down side in photo) but except this, spot was good.

3. during hybridization, blocking agent is BAS and salmon sperm DNA, but it blocks unspecific bong on glass. then we cannot bloxk unspecific DNA bond, such as probe bind with DNA beside the mark?