Hello Friends,
I am facing a problem.. I recently started with a project where I got to amplify gene of about 600 bp. Some other fellow was working on this before and he already had forward and reverse primers...
I was trying to amplify the gene using PCR. However, each time I received a smear.. I tried several conditions.. but it wont work...
When I analysed the primers, I found that there is a 5 base mismatch at the 3' end of the forward primer.. Do you think it's a major problem? Should I redesign the primer?
I would highly appreciate any help!
Regards,
Bhaskar
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5 replies to this topic
#2
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Posted 20 September 2004 - 09:13 PM
Is the mismatch at the very 3' end (the last nt)? If yes, you may have to redesign a primer. Once I missed a nucleotide at the 2nd last position of my forward primer and it still worked.
#3
kant0008
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Posted 20 September 2004 - 10:34 PM
Hi,
5 bases at the end of a primer sounds like a lot, I wouldn't think it would work. Depends how long it is as well. Do you know whether it worked well for your collegue?
5 bases at the end of a primer sounds like a lot, I wouldn't think it would work. Depends how long it is as well. Do you know whether it worked well for your collegue?
#4
bhaskar_dutta
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Posted 21 September 2004 - 01:16 AM
Thank you twister and kant0008 for the reply.
The reaction didn't work anytime before.. but it does give a smear so i was trying my best before I rethink to design the primer again...
Thanx one again..
Bhaskar
The reaction didn't work anytime before.. but it does give a smear so i was trying my best before I rethink to design the primer again...
Thanx one again..
Bhaskar
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Posted 21 September 2004 - 09:08 AM
Oh, My, I didn't notice there are FIVE mismatches. In that case, you should redesign it.
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smesme
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Posted 23 September 2004 - 12:42 AM
Hi Bhaskar,
yes you should definitely redesign.
Generally, the 3'end where the polymerase starts the elongation is the most important site, regarding specificity. Actually, allele-specific PCR which is used for SNP detection utilises 2 different primers which only differ in the 3'end as suifficient discrimination. So this is very important!
regards, Søren
Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark
www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes
yes you should definitely redesign.
Generally, the 3'end where the polymerase starts the elongation is the most important site, regarding specificity. Actually, allele-specific PCR which is used for SNP detection utilises 2 different primers which only differ in the 3'end as suifficient discrimination. So this is very important!
regards, Søren
Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark
www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes
