3 replies to this topic

[FabBor]

Hello Friends!

I am working with DNA extractions from Plants in order to amplify Geminiviruses (GV).
I am trying to amplify the top part of the A component.

My samples are fresh, we stored the matherial at -70C
My PCR solutions are fresh too and we do store them at -20C

The weird thing is that when I do the PCR for this region (Top-A) with the primers known as 715 and 1978 I get only a beautiful smear in my gels. And the extraction control (a positive sample that we use to control our extraction method) shows the smear too but it amplifies (it shows the band in the middle of the smear). The PCR product expected is around 1500bp.

I did a Dot Blot hybridization with my samples with a general probe for GV and I do know that my samples are positive for GV (The Dot was not too strog, but I was able to see it, an in other samples is was barely observable but was there). Even more, I have obtained PCR products when I try to amplify the Hypervariable region (HV) with a PCR expected product of 300bp.

How come my samples do not amplify!!???  We even ordered a new primer set and It did not work!

I have changed the annealing temperature, I have made new extractions, I asked a Friend in the lab to do new extractions for me (Karma hypothesis) and I have changed every single PCR solution and still I get only smear, beautiful smear.

Could some one please give me any light with this issue!!!

Thank you very much in advance.

A desperate Molecular Biologist

[jiajia1987]

FabBor, on Mar 2 2009, 09:46 AM, said:

Hello Friends!

I am working with DNA extractions from Plants in order to amplify Geminiviruses (GV).
I am trying to amplify the top part of the A component.

My samples are fresh, we stored the matherial at -70C
My PCR solutions are fresh too and we do store them at -20C

The weird thing is that when I do the PCR for this region (Top-A) with the primers known as 715 and 1978 I get only a beautiful smear in my gels. And the extraction control (a positive sample that we use to control our extraction method) shows the smear too but it amplifies (it shows the band in the middle of the smear). The PCR product expected is around 1500bp.

I did a Dot Blot hybridization with my samples with a general probe for GV and I do know that my samples are positive for GV (The Dot was not too strog, but I was able to see it, an in other samples is was barely observable but was there). Even more, I have obtained PCR products when I try to amplify the Hypervariable region (HV) with a PCR expected product of 300bp.

How come my samples do not amplify!!???  We even ordered a new primer set and It did not work!

I have changed the annealing temperature, I have made new extractions, I asked a Friend in the lab to do new extractions for me (Karma hypothesis) and I have changed every single PCR solution and still I get only smear, beautiful smear.

Could some one please give me any light with this issue!!!

Thank you very much in advance.

A desperate Molecular Biologist

hello there,

i am currently having this irritating problem too.

i have done a few PCR and each time i am doing this particular one (one for which i am generating a library by amplifying it), i always get a smear instead of the amplified product. the funny thing is that i don't get smears for my other PCRs! i am redoing it today by increasing the extension time and doing a dilution of the templates before adding it into the PCR mix. you may wanna try that. hopefully, this problem goes away.

[NemomeN]

How did you prepare the DNA?

[phage434]

It's hard to know without a lot more detail, but a wild guess is that you have far too much genomic DNA template in the PCR reaction.  Try diluting the DNA 100x and 1000x and retrying the PCR.