Hi Mary,

I don't use a tool to design primers but I always test them in Netprimer to see whether they are ok. You can find Netprimer here:

http://www.premierbiosoft.com/netprimer/

If you want to use the primers for RT-PCR, I would go for a Tm of around 65-70 ˚C and they should have no predicted secondary structure (Netprimer predicts e.g. hairpin or dimer formation). On their 3' end it is best if they terminate with two or three Cs or Gs. I recommend you use primer pairs, in which the two primers target different exons, so that at least one intron lies between them. This way you can distinguish whether your amplification is coming from cDNA or from contaminating genomic DNA. For specificity you can use Blast as described above.

You can check this software on line http://frodo.wi.mit.edu/ but you need to have your template sequence....

You can use the option -> primer blast in ncbi website http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome

Hi Mary,

I don't use a tool to design primers but I always test them in Netprimer to see whether they are ok. You can find Netprimer here:

http://www.premierbiosoft.com/netprimer/

If you want to use the primers for RT-PCR, I would go for a Tm of around 65-70 ˚C and they should have no predicted secondary structure (Netprimer predicts e.g. hairpin or dimer formation). On their 3' end it is best if they terminate with two or three Cs or Gs. I recommend you use primer pairs, in which the two primers target different exons, so that at least one intron lies between them. This way you can distinguish whether your amplification is coming from cDNA or from contaminating genomic DNA. For specificity you can use Blast as described above.

You cannot use PCR to isolate truly unknown genes as you need DNA to design your primers from. In order to isolate up-regulation of genes in response to something, you might want to use a microarray, RNAseq, or proteomics assays.

If you mean genes that are annotated but have no known function, you can use any number of different primer design softwares or design them yourself. There are guides online but the basics of primer design are the following:

40-60% GC content

Tm between 55 and 60

have a G or C at the ends of the primer

Make sure there is no secondary structure in you primers

Make sure there are no primer-dimers in your structure

Use NCBI's primer blast to make sure your primers are specific to your region of the genome.

To do most of my analysis for primers I have designed, I use oligo analyzer on the IDT website.

http://www.idtdna.com/analyzer/applications/oligoanalyzer/

Thanks for the replies. I was able to design the required primers.

Hi all,

I am currently conducting a research on the primer design as many LAMP users find it difficult to get their way around getting a validated and optimised functional primer.

I was wondering if their are any assay that are any assay that are optimised and validated ready to use assays/primers for specific target. would it not safe time and add to the convenience besides getting a functional primer ?....

Use insilico PCR