question about Northern-blotting probe design - (May/22/2007 )
I'd design a northernblotting probe using pcr products following TA cloning, but this pcr product (250bp) contains 40bp of intron sequence at up and down-stream, respectively, my questions are 1)if this probe will work? 2) if this non exon sequence will affect the sensitivity of Northern blotting. Hope experienced experts give some suggestions. greatly appreciated!
I don't think I am an expert per se... I have done northerns, but I never tried using genomic sequence in the probe... why not just get rid of the extra sequence... cut it out with RE or something... The extra sequences may or may not affect the northern sensitivity and specificity, depends on what the sequence is, blast may give you an idea of if it will work or not... probably only choice is to determine empirically or do a little more work to get the correct probe sequence in the first place...
HTH and good luck to you!
yea i am also not an expart but have some experience. but always i made probe from coding region. if you can design probe without the intron ........that will be good i think. if you want to just detect something then probe length will not matter, but if in you case probe length is important then you need to think more
Thanks a lot for your suggestion. I think you guys are right, a cautious beginning may bring good result. I was kind of lazy and tried to use available things on my hand. Sounds like I have to make a new design.
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