hi there,
a very unusual question which crossed my work these days. I designed two primers for a PCR which turned out to not working. When I checked the primers against the genome I found out that they are very specific BUT that the gene is positioned on a minus strand (complement). May this be the problem that I designed my primers for a plus strand and that they should be complemented?
thanks for nice replies!
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#2
phage434
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Posted 24 January 2011 - 06:02 AM
No, PCR and DNA copying in general is independent of the orientation of genes. PCR enzymes have no bias for gene direction. Your problem is elsewhere.
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wilson_trace
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Posted 05 March 2011 - 01:40 AM
tatzilo, on 24 January 2011 - 02:35 AM, said:
hi there,
a very unusual question which crossed my work these days. I designed two primers for a PCR which turned out to not working. When I checked the primers against the genome I found out that they are very specific BUT that the gene is positioned on a minus strand (complement). May this be the problem that I designed my primers for a plus strand and that they should be complemented?
thanks for nice replies!
a very unusual question which crossed my work these days. I designed two primers for a PCR which turned out to not working. When I checked the primers against the genome I found out that they are very specific BUT that the gene is positioned on a minus strand (complement). May this be the problem that I designed my primers for a plus strand and that they should be complemented?
thanks for nice replies!
Hello,
A primer pair is designed after considering both the sequence strands, the sense and the anti-sense. In my opinion, you may check the annealing temperatures of the designed primers. It might be possible that the presence of some secondary structures is hindering the hybridization of the primers. You may also check for primer secondary structures such as self dimers, hairpins to see if they are available for hybridization.
I usually use Primer Premier for designing primers for my assays. Visit: http://www.premierbi...sign/index.html
Wilson
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wilson_trace
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Posted 05 March 2011 - 01:44 AM
tatzilo, on 24 January 2011 - 02:35 AM, said:
hi there,
a very unusual question which crossed my work these days. I designed two primers for a PCR which turned out to not working. When I checked the primers against the genome I found out that they are very specific BUT that the gene is positioned on a minus strand (complement). May this be the problem that I designed my primers for a plus strand and that they should be complemented?
thanks for nice replies!
a very unusual question which crossed my work these days. I designed two primers for a PCR which turned out to not working. When I checked the primers against the genome I found out that they are very specific BUT that the gene is positioned on a minus strand (complement). May this be the problem that I designed my primers for a plus strand and that they should be complemented?
thanks for nice replies!
Hello,
A primer pair is designed after considering both the sequence strands, the sense and the anti-sense. In my opinion, you may check the annealing temperatures of the designed primers. It might be possible that the presence of some secondary structures is hindering the hybridization of the primers. You may also check for primer secondary structures such as self dimers, hairpins to see if they are available for hybridization.
I usually use Primer Premier for designing primers for my assays. Visit: http://www.premierbi...sign/index.html
Wilson
