Primer design - How to - am a novice ? (Jul/16/2008 )

Hi all , I need an answer to this urgently .
I have been asked to design primers for my gene but I have never done this before , i looked up some softwares online but i don't understand anything . Can somebody explain how to go about this.
Thanks a lot .

---

1. what's the region of interest
2. search the gene sequence (pubmed or other similar website). I made a document with all the sequence and highlight the exons.
3. serch the region of interest in the sequence and copy/paste it in one of the primer design software or tool) (I use primer3).
4. after getting the best primer design use blast nucleotide (pubmed) to check specificity of the sequence.

---

Thanks , it sounds easier now . Though i have one more question - what do i do if the gene has isoforms?

---

There are several alternatives. Here are a couple:
If the isoform of interest is tissue-specific, you need to extract mRNA from that tissue and do some RT-PCR to get your isoform as cDNA.
If the isoform is one of several expressed in one tissue, you might as well just go from genomic DNA, and amplify what you can then screen. The only simple way out is if the isoforms differ at their 5' or 3' ends. In that case, make one primer specific to your desired isoform (typically by having the last few bases specific to your target), and it should be the only one that amplifies.

There are probably a few other choices out there.

---

where can I download primer 3? is it expensive or is it free?

---

[ where can I download primer 3? is it expensive or is it free?
[/quote]


It is an online free software programe, you can access it by clicking the following link:
http://biotools.umassmed.edu/bioapps/primer3_www.cgi

---

But what about tetra-primer ARMS-PCR design? anybody knows a program? becase Im trying to acces the program described by Shu Ye et al in 2001, but the site dosen't work anymore

---