3 replies to this topic

[Tatiana]

Hi, I am trying to amplify an intron using genomic DNA as template. We have the primers and we know that they work because we tested them with other templates. Also, we tested the genomic DNA using tubuline and actin primers and we saw the bands. The problem is that when we used both the primers and the genomic, we do not have anything, no bands. We did Mg curves, gradient curves, put either formamide, glycerol or DMSO in the reaction mix, and we do not see bands, we do not amplify anything. Until now we used three different polymerases: regular taq from Sigma, other for long PCR from Roche and the redtaq from Sigma too. Do you have other suggestions? I really need your help because I tested many things and now I am impatient to solve this problem   .

Thanks a lot!

Tatiana

[labrat]

Hi Tatiana,

You must have known the size of your desired product, right, then how big is that? If it is too long, amplification could be problematic. Could be there some genetic alterations in your cell such as chromosome deletion? just wondering.

[Tatiana]

Tatiana, on Sep 15 2004, 06:05 PM, said:

Hi, I am trying to amplify an intron using genomic DNA as template. We have the primers and we know that they work because we tested them with other templates. Also, we tested the genomic DNA using tubuline and actin primers and we saw the bands. The problem is that when we used both the primers and the genomic, we do not have anything, no bands. We did Mg curves, gradient curves, put either formamide, glycerol or DMSO in the reaction mix, and we do not see bands, we do not amplify anything. Until now we used three different polymerases: regular taq from Sigma, other for long PCR from Roche and the redtaq from Sigma too. Do you have other suggestions? I really need your help because I tested many things and now I am impatient to solve this problem  .

Thanks a lot!

Tatiana

Hi,

Thanks for the answer. By similarity with other specie we calculated that the intron is 2200 bases, is not so big. About the deletions, I do not think our animals have it. Do you have any recommendation?

Thanks again!

[smesme]

Hi Tatiana,

what template did you test the primer on so you know they work?If your primers are in the coding regions, you could try adding some cDNA to the same mastermix so you know that it works and that the gene is there.

If the intron has very high GC content, amplification can be a problem. One way could be to try a higher denaturation temp. (97 C) and add a little exstra TaqPol.

Regards, Søren

Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark

www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes