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Chooseing the best sequence for clonning - How to choose the best sequence for clonning a cDNA (Aug/16/2009 )

Hi there!

I'm trying to clone a cDNA fragment, but I can't amplify it!

I double checked my primers, tested 2 diferent cDNA libraries (beta actin OK) and nothing. I designed the primers to amplify the CDS and added the adaptors for restriction enzymes.

I was wondering if my primers were properly designed , since i used a sequence discribed as : Equs caballus (NAME OF THE PROTEIN), mRNA.

NCBI site describes an exon in the region of my foward primer. Could this be a problem (considering is a mRNA sequence)?

Thank you all!

-Ivanov_br-

* Check the primers for primer/dimers, hairpins with 5' overhangs. Use the IDT tools (idtdna.com).
* gradient PCR to get the correct Tm
* try dilutions of your template DNA to make sure you don't have inhibitors

* next: very important. Redesign your primers. Do this after 3 days, not 3 weeks. Use Primer3.
* order several primers to amplify smaller regions of your DNA, especially with the primers in highly conserved regions
* order some primers without the 5' overhangs you really want, to make sure they are not causing your problems.

-phage434-

thank you for the tips, Phage434.

I've tryed the Tm - 5C, and at the exact Tm. Should I test at higher temperatures or lower?

Thanks again!

-Ivanov_br-

I always use 55C for my new primers. It almost always works if your primers are designed reasonably.

-phage434-

Thanks again, Phage.

Now it workd. I used 55C and added 2% DMSO. In addition, I tested 1.5 mM, 2.0 mM, 2.5 mM and 3.0 mM MgCl2.

2.5 mM and 3.0 mM worked very well!

Thanks again for the help!
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-Ivanov_br-

I think people get in trouble messing with the Mg++ concentration. I'd suggest a premix. You have many variables in a PCR reaction: quality and amount of template, primer sequences, cycling conditions. The last thing you need is a few more knobs to turn, most of which will break things.

-phage434-

In fact, this was my first problem with PCR. I've always used 1.5 mM Mg++ in my reactions. Since I figured that primer dimers could be a major problem in my reaction, I tested different Mg++ concentrations.

Anyway, a premix is very usefull.

-Ivanov_br-