designing genome specific primers

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designing genome specific primers

Postby Bobby89 » Sep 09 2014 6:48 am

I am having problems with amplification of approx. 1kb regions flanking a gene. i am using gDNA as template but i keep getting unspecific products. i am fairly sure my problem is due to bad primers. i have tried optimisation for a week now, so it is a waste of ยงยง, which is why i will order new ones..
The genome i am working with is sequenced (which i have) but it is not on NCBI so i can use primer blast.. Is it possible to design two primers within fairly short span of each other (3kb) so that they are still fairly unique to the genome ?

basically i am amplifying the 3kb region to work with further down the line, so i only need one working and preferable fairly specific pair of primers,
please note that i don't have access to commercial software packages.

can someone explain how to go about designing genome specific primers in this case ? preferably in a step by step.

Thanks
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Re: designing genome specific primers

Postby mchlbrmn » Sep 10 2014 10:32 am

Is your Tm calculation method set for the correct Mg and salts concentration, and compatible with the enzyme and buffer?

It may be a good idea to design several sets of primers and see which works best. I prefer to try more than one set for genomic DNA even before I encounter problems, since genomic is more difficult.

Is it possible to design two primers within fairly short span of each other (3kb) so that they are still fairly unique to the genome ?
I think the proximity of the primers is generally unrelated to their uniqueness in the genome, unless the gene is duplicated.
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Re: designing genome specific primers

Postby stubbedstarbucks424 » Oct 22 2016 5:36 am

mchlbrmn wrote:
It may be a good idea to design several sets of primers and see which works best. I prefer to try more than one set for genomic DNA even before I encounter problems, since genomic is more difficult.

I think the proximity of the primers is generally unrelated to their uniqueness in the genome, unless the gene is duplicated.


Liked the idea
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