Need advice in primer designing

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aaroncjj_2020
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Need advice in primer designing

Post by aaroncjj_2020 » Sat Jan 13, 2007 1:10 pm

dear all

i've an unpublish sequence of the coat protein of banana mild mosaic virus. i hv tried some primers but it doesnt give me the sequence that i wan. hope to get some advice in dsigning primers for my sequence.

thanks a lot.

oppox
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Post by oppox » Sat Jan 13, 2007 5:53 pm

its hard to know where to start without seeing the gene. the forward primer should be located in the beginning and the reverse in te end of the gene. thats pretty much it.
Its hard to say anything but that right know since I dont know what u are going to do with it, are u having primers with a restriction site?
what sytem are u going to put it in?
also u can check up on touchdown PCR if u get unspecific binding.
If your primers contain to much AT u can add some GC.

lupine_nickt
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Post by lupine_nickt » Mon Jan 22, 2007 2:31 pm

Keep them a similar m.w. & length; at least 15 b.p., probably more (but not too many - you're not after a megaprimer, after all); try to end the primers on a G/C.

That's about it, really.

xF,

...Nick

SororSaudade
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Post by SororSaudade » Mon Jan 22, 2007 4:42 pm

have you tried different annealing temperatures?

hopefully this paper will help you as it did to me:

http://bioinformatics.oxfordjournals.or ... t/bti633v1

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