This depends on whether you are amplifying your PCR product from gDNA or cDNA, and where your primers sit in the gene. If you designed your primers to sit in an intron, you won't be able to amplify from cDNA. If they are exonic or in the UTR, you will be able to amplify from cDNA but the product will be smaller than gDNA because the introns will be spliced out. If you are amplifying from gDNA, you have no worries.

I recommend designing your PCR primers from the same template that you wish to amplify from. If you are going to use gDNA, design them from genomic sequencing. If you are going to use cDNA, design them from the mRNA sequence.

This depends on whether you are amplifying your PCR product from gDNA or cDNA, and where your primers sit in the gene. If you designed your primers to sit in an intron, you won't be able to amplify from cDNA. If they are exonic or in the UTR, you will be able to amplify from cDNA but the product will be smaller than gDNA because the introns will be spliced out. If you are amplifying from gDNA, you have no worries.

I recommend designing your PCR primers from the same template that you wish to amplify from. If you are going to use gDNA, design them from genomic sequencing. If you are going to use cDNA, design them from the mRNA sequence.

Agree with Michael Green on possible effects, depending on the positions of your primers in the gene sequence.

Though I disagree slightly that you should design PCR primers using the same template that you will amplify from. Sometimes this is not possible due to information not available. It should be fine to design qPCR primers from genomic sequence, for example, as long as you are clear on the intron and exon positions.

Good point Jun. I work on humans and mice, so I have all of the information that I need, but I acknowledge this is not always the case when you are working with other species.

Thank you guys so much. Micheal my Primers are designed from gDNA, albeit I will have to consult my supervisor if they're designed from introns. Another thing that worries me is, could there be a possibility that I'm using high amount of primers ? I mean apparently dimers are quite visible in every result. Also the Tm for my Forward and reverse primer is 75 and 72 respectively. Where as The maximum annealing temperature that "according to my supervisor" I can set is 56. I don't get this, in theory what I know is that we can set annealing temperature by subtracting 3-5 from Tm. So why does my teacher insist that I can't go any higher than 56 as annealing temperature :/

Hi Ghina. It sounds like you need to re-design your primers. They need to be designed to avoid primer dimer and hairpins. The Tms are also much too high, which suggests that either your primers are too long or the GC content is too high. I usually design my primers to have Tms of 60, and this is pretty standard.

Here is a good tool for designing primers:

http://tools.lifetechnologies.com/content.cfm?pageid=9716

You can check whether you're going to have primer dimers using this tool: http://www.thermoscientificbio.com/webtools/multipleprimer/

With respect to primer concentration, I usually keep my freezer stocks at 100uM. Then I use a 1:10 dilution for my working stock (10uM), and use 1uL in a 20uL PCR reaction. Here is a tool you can use to calculate how much water/TE to add to obtain a certain concentration: http://www.idtdna.com/Calc/resuspension/

Dear Micheal,

The primers are designed from exon region and the GC content is not too high, may be I forgot to mention these are for the purpose of gateway cloning. The gene was isolated from Sea buck thorn but has a consensus with that of tomato. The primers are also designed from the consensus region (exon) of both tomato and sea buck thorn. I will check the links that you suggested and see where it takes me. Thank You so much.

Regards.