What's the difference between the lanes with primer-dimer and the lanes with the broad range smears? To me this looks like your primers aren't working as they should.
I'd suggest,
1) if you haven't already, blasting your primers to ensure that they are a monospecific in the Dictos genome and, if the resource is out there, ensure that they aren't on top of any known SNP or CNV.
2) Include a different working PCR reaction and an NTC so you can exclude the PCR reagents/reactions as the problem.
3) Look at your reaction conditions if you have a gradient cycler start with a temp curve and Mg profile.
4) As Cathal suggested, try a different polymerase
Zeb
Date: Wed, 16 May 2012 23:21:02 -0700
From: jitesh.green@gmail.com
To: diybio@googlegroups.com
Subject: Re: [DIYbio] facing problem with pcr gel results
its dictyostellium genome... finally resuspended in 1:5 TE buffer.
On Wednesday, May 16, 2012 4:53:11 AM UTC+5:30, phillyj wrote:
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I'd suggest,
1) if you haven't already, blasting your primers to ensure that they are a monospecific in the Dictos genome and, if the resource is out there, ensure that they aren't on top of any known SNP or CNV.
2) Include a different working PCR reaction and an NTC so you can exclude the PCR reagents/reactions as the problem.
3) Look at your reaction conditions if you have a gradient cycler start with a temp curve and Mg profile.
4) As Cathal suggested, try a different polymerase
Zeb
Date: Wed, 16 May 2012 23:21:02 -0700
From: jitesh.green@gmail.com
To: diybio@googlegroups.com
Subject: Re: [DIYbio] facing problem with pcr gel results
its dictyostellium genome... finally resuspended in 1:5 TE buffer.
On Wednesday, May 16, 2012 4:53:11 AM UTC+5:30, phillyj wrote:
I don't know where your gDNA is coming from, but maybe you got some DNAse in it? What is this sample from? How was it collected/stored?On Tue, May 15, 2012 at 1:47 PM, Nathan McCorkle <nmz787@gmail.com> wrote:
can you label the gel lanes, other than the MW ladder I'm not sure what to make of anything--On Tue, May 15, 2012 at 6:53 AM, jizzs <jitesh.green@gmail.com> wrote:
i have been running pcr gel for a 1.3kb gene but getting nothing except long smears with no primer dimers at end .
i dnt know what is the problem ......
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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
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