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plasmid amplification - exons and primers (Apr/22/2006 )

Good day to all!
I have problems when i used a primer that spans exon 4/5/6 to amplify plasmid DNA containing exon 5 and 9 of my target gene (the plasmids were prepared somewhere else). There is no amplification detected at all. Isn't it suppose to amplify at least that exon 5? By the way, I am using SYBR Green for detection. We know that Sybr Green is the most sensitive (even less specific). In the mean time we are also trying to design primer that span exon 9 of this gene.

Please help. Thankssss. sad.gif


wait a minute, let me see if I'm understanding the problem...

you have primers that bind like this:

xxxxxxxx(fwd) --><-- (rev)xxxxxxx

your clone is like this:

is this right?

because, then, of course you will not see a product when you amplify from the clone. you do not have the sequence for the second primer to bind. does this make sense? also, what size is your expected amplicon? if you want to see good amplifcation from your clone (if I have diagrammed this construct correctly) then you will need primers like this:


did I help? and the format was funky when I typed it...assume the primers do not directly abut each other cool.gif