Re: [DIYbio] Designing primers for PCR

On Fri, Feb 8, 2013 at 12:47 AM, Mega <masterstorm123@gmail.com> wrote:
> Hi everyone,
>
>
> I'm trying to design primers on my own. but now there are a few questions
> that Google couldn't answer me.
>

Do you know how to use Python? You should probably start messing with
bioPython.... You can spin your own primer design code that way

Here's a good quick overview of the things you need to watch for
during primer design
http://instructional1.calstatela.edu/jmomand2/2004/programming/ppt/chan_ronny.ppt
it says:
Primer length
Melting Temperature
GC content
Hair-pin loop
Self-dimerization
Cross-dimerization

The main thing you need to look at is melting temp calculations:
http://biopython.org/DIST/docs/api/Bio.SeqUtils.MeltingTemp-module.html#Tm_staluc

and this code extends the nearest neighbor Tm calculation with
monovalent ion, Mg2+, dNTP, DNA, and DMSO corrections (references in
the code)
http://pastebin.com/PijkvZr5

here are some primer tools that may or may not be good, I haven't
looked through the code to see, and I haven't tested it:
http://sourceforge.net/projects/pythia/

This bioPython introduction has tutorials on how to use it with
Primer3 (starting around 2/3rd the way through the PDF), all right out
of the box:
http://www.biopython.org/DIST/docs/presentations/biopython.pdf

--
-Nathan

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