I met a problem on ISH, I synthesized a long chain composed of a part of U6 snoRNA sequence (5’-TTCACGAATTTGCGTGTCAT) and another 2 parts to bind my components, to act as a positive control. but i cannot get a screen full of blue dot-the precipitation of BCIP and NBT that could interact with AP to visualize the signal. I check all components and reagents and renew them , got the better result but still can’t get my ideal result. So i think maybe the hyb temp is the point. I first use the recommended temp-57dgr, bad result. So i increase it to 65 dgr, better but still can’t fulfill the screen. I calculate the Tm again but still can not figure out why. So is anyone any suggestions on the temp? The sequence the hybrid is mentioned above. Thank you~
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