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dobrui dobrui
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12 years ago
Hi All,
I am having the following problem. I work with a fusion gene and need to do a QPCR. I found a couple of papers with primers I can use but have no idea how people manage to design those primers and what is the product length etc.

therefore I would like to ask you guys if anyone is aware of how to design qpcr primers for a fusion gene. Also, it needs to pick up different isoforms of the two fusion partners.

Please let me know if you have some thoughts on that, some ideas of software or website.

So far, I tried NCBI primer design, primer 3 and thinks like that but they all need input sequence of the gene. I can find out the sequences for each of the fusion partner, I can find the 20nt sequence where they fuse together, but I need automated way to screen for where exactly this sequence overlapps with the 5' and 3' partner.

sorry for the long post, I hope you got an idea of my problem and could advise.

thanks in advance
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wrote...
12 years ago
I use the Harvard Primer Bank for all of my qPCR primers.

http://pga.mgh.harvard.edu/primerbank/

I normally order 2-3 sets of primers and then test them for similar amplification efficiency with my control primers and also check for single product with a dissociation curve.

If you're not doing mouse or human qPCR try Perl Primer

http://perlprimer.sourceforge.net/

You can blast the resulting primers to make sure they are unique. Also always do dissociation curve on new primers (or run your products on a gel) to make sure they're truly unique.

All this is free btw.

Good luck! Let us know what you come up with.
wrote...
12 years ago
Read this article Wink Face How to Choose Primers for qPCR
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