5' RACE - gene specific primer - (Mar/21/2006 )

i will do 5' race pcr with my gene of interest. here in my lab is a roche race pcr kit.
i've some questions concerning the primer i have to create.

its recommended that one should synthesize cdna by using a gene specific primer.
that means that my primer has to prime with the mRNA of my gene, right?

then only cdna of my gene should be synthesized, right?

then i have to do nested-pcr with two another gene specific primer. but these two primers must prime with the cdna, right?


so how to create a primer against the mrna? just translating gdna into mrna and then create? i am a little bit confused of that, can someone give me an example?

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Hi

Yes, but in my opinion it is not necessary. I did a lot of 5´-Race with GSPs and Universal primed cDNA and saw no difference.


Let´s say: Most of your cDNA should be your gene of interest. In my experiments i could still amplify housekeeping genes out of specific primed cDNA.


Yes


You should know your mRNA sequence and then you can easily design your primers just like you are doing it for standard PCR.

But be warned. Maybe you will see nothing on your gel after your first PCR. But this shouldn´t bother you. Just perform a nested PCR.
I would suggest that you use a Primer with high Tm for your first PCR and use high annealing temperatures.
And I would highly recommend that you place your primer near the expected 5´ end. This would enhance your efficiency a lot. But on the other hand, if you are working in a eucaryotic system, it is preferred to place your primer not in the first exon, so that you can be sure you did amplify cDNA (no intron present) and not genomic contamination. If this is not possible or if you deal with procaryotes, then you have to include a negative control (cDNA sythesis with no RT added) to ensure that you have no genomic contamination.

Good luck

Morrison

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