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Old 10-26-2016, 10:33 AM   #1
danova
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Default PCR amplicons primer design - need advice

Hello,
I have a list of dna sequences corresponding to a family of HSP proteins in Bacteria (800 seqs of length average 570nt). I would like to design PCR primers for miseq amplification (2*250pb).

The insert size should be around 460bp since this is the length that is good quality from the miseq sequencing to be analysed.

I was wondering if you can suggest a pipeline and tools to design my PCR primers that cover most possible species, which are likely to be degenerated.

My first idea to create an alignment for all sequences using Muscle.

From there how do i get degenerated primers ?

Thanks for your comments.

Last edited by danova; 10-26-2016 at 11:14 AM.
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Old 10-27-2016, 05:33 AM   #2
thermophile
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are you looking for snp's? if so you should be designing your primers to hit a smaller target because you want a lot of overlap in your sequences. Last time I designed primers (it was a while ago) I used ARB-aligned my seqs and used the primer design tool to get potential targets.

The above is assuming that you've checked lit for other primers hitting your target gene, compiled a list of those, tested in silico against your seqs, and found none appropriate.
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Last edited by thermophile; 10-27-2016 at 05:35 AM.
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Old 10-27-2016, 07:23 AM   #3
danova
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Not looking for SNP , just the HSp proteins that are very conserved across species.
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