primer design - (Nov/23/2009 )

wat r the best software to design a primer?

hazrina on Nov 22 2009, 11:21 PM said:

maybe u can try using primer pemier 5

hazrina on Nov 23 2009, 01:51 PM said:

hi,
i use primer3 software to design mine...

DRN on Nov 23 2009, 08:59 AM said:

it is possible to design primer manually?coz i couldn't fine good primer in the location that i want..primer that i get from primer 3 or other tools only found primer located from 5oobp...it was to far for me to work reverse...this is how i want the primer to be design..


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my dna was about 1.5 kb..it was located at the centre of the gene...i need to find in front and the back of it to find promoter and also the polyatail...

hazrina on Nov 24 2009, 09:25 AM said:

yeah, you can do it manually as well....in that case you will have to determine the Tm of your primers (which should in turn be optimized depending on the GC/AT richness of the DNA of your interest and also BLAST it to check if its picking up any non-specific DNA also....

If you are willing to take some risk, then yes, eyeballing primers works quite well. Find a region with (ideally) 50% gc. Aim for a 20 bp region ending in one or two GC's. Avoid primers with more than four 3' GC's in a row. If your sequence has high AT, make the primer longer, perhaps 24 bp. If high GC, make it 18 bp. Check the primer pair with the tools at idtdna.com for primer hairpins (only the 5' overhangs with a strong 3' end match matter) and primer dimers. I wouldn't bother to calculate Tm, it is over-rated.

Dear Haz,
If i not mistaken you are still searching your promoter and poly-a-tail...
to design a primer you got to have both before you can clone the whole gene out.
If you want to design primers to find promoter and poly-a-tail, I would think such way is hardly possible when you don't even have the full genomic sequence or the species or related species.

I use primer 5, but I think you can use primerBLAST in NCBi. it is very good.

For making full length cDNA try RACE (rapid amplification of cDNA ends) kits - I think Roche and a few other companies have them.

I used BLAST...