primer-blast from ncbi - (Nov/24/2009 )

Has anyone use this software before?

I am new in pcr primer design and want to design primers for rt-pcr. This software has involve chossing of exon-exon junction for users so i thinking to use it. But, how does it compared with primer3? which is better?

thanks

wntiong on Nov 24 2009, 05:25 PM said:

I think primer-blast is the best site around these days. It implements the original Primer3 algorithm. However, one difference is the annealing temperature calculation, while Primer3 was always a bit too high (you'd have to input 62C to get a primer for 60C), Primer-Blast is correct. Recommended!

Cheers,
Minna

wntiong on Nov 24 2009, 09:25 PM said:

For me, I used PerlPrimer software to design the primer for real-time. You can google it to find the download link.

Thanks guys,

i used both primer3 and primer-blast to design primer for same gene, then blast both forward and reverse primer sequences. Apparently two different set of primers were produced and after blast, the primer3's primers showed e value of 0.0015 to that gene's genomic sequence and about e value = 248 for the next genes. for primer-blast's primers showed same e value of 0.0015, but if against to next other gene, it is about 3.5 of e value.

i am not sure. but since the primer-blast has the option to find the exon-exon boundary, which is better according to above blast results?

i am really a beginner onto this, kindly advise though if the question sounds stupid. lol.

thanks