In-situ Hybridization RNA probe - wrong size? (Jan/07/2008 )

Hi all,

I have in-vitro synthesised a DIG labelled-RNA probe using SP6 RNA polymerase and run gel to verify the probe size. Instead of a very faint band at the product size of ~300 bp, I obtained another two bands of ~700 bp and ~3kb.

I was told by senior that the ~3 kb band is my vector (I was using pGEM-T vector). Why I still can see my vector after DNAse treatment? I think DNase treatment suppose to remove all the double-stranded DNA?

Besides, can anyone let me know why I get the extra ~700 bp products? Both the ~3 kb and ~700 bp products are much more intense than my expected product of ~300 bp.

Can i still use it for ISH? I was told by friend that as long as u obtain bands in the gel, it can be used for ISH despite the size, this make me confuse. Pls help.



Kuah

Is this a denaturing gel? You may be seeing secondary structure or dimerization of your probe if not.

no, i just used normal agarose gel. I heated up the sample at 60C for 10 min, on ice 2 min before loading. My RNA markers was seperated nicely using the same way. Anyway, I will try to prepare a denaturing gel.