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No Amplification -- Primer Dimer

No Amplification -- Primer Dimer - PCR - Polymerase Chain Reaction Forum

No Amplification -- Primer Dimer - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #11  
Old 09-09-2011, 06:29 AM
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Default Re: No Amplification -- Primer Dimer

And also another doubt Sir. How to confirm that the bacterial genomic DNA is fine... Or denatured???
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  #12  
Old 09-19-2011, 04:48 AM
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Default Re: No Amplification -- Primer Dimer

I changed the PCR cycle... And I didn't get the primer dimer this time... Is the change in the cycle cause the dimer to disappear???
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Old 09-19-2011, 10:22 AM
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Default Re: No Amplification -- Primer Dimer

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Originally Posted by saranyah View Post
And also another doubt Sir. How to confirm that the bacterial genomic DNA is fine... Or denatured???
To check Bacterial DNA you can run it in the gel. If you see clear band at your expected size then it is ok. If you see smear or if it stuck in the well then there is degradation or high salt etc.
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  #14  
Old 09-19-2011, 10:23 AM
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Default Re: No Amplification -- Primer Dimer

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I changed the PCR cycle... And I didn't get the primer dimer this time... Is the change in the cycle cause the dimer to disappear???

Did you get the pcr product or not. and what you changed in the pcr cycle
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  #15  
Old 09-20-2011, 01:55 AM
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Default Re: No Amplification -- Primer Dimer

I didn't get the amplification.... And also no primer dimer.... The genomic DNA was checked for 16S rRNA universal primer and it showed a very good amplification... But I'm still not getting my gene out of it... I'm frustrated... Its been a month Sir...
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  #16  
Old 09-20-2011, 09:37 AM
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Default Re: No Amplification -- Primer Dimer

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Originally Posted by saranyah View Post
I didn't get the amplification.... And also no primer dimer.... The genomic DNA was checked for 16S rRNA universal primer and it showed a very good amplification... But I'm still not getting my gene out of it... I'm frustrated... Its been a month Sir...
Hi Saranya,

There is no need for frustration. No protocol, No experiment works perfectly in first time,if ie the case then every one will finish there project in a week.
Once i took more than 3 month to optimize my PCR protocol.

Please write what exactly are you doing. some time you are writing bacterial DNA, then now genomic DNA. If you write each details your pcr protocol, components, Template source, expected size of your product etc etc. It will help to answer your question correctly.
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  #17  
Old 09-20-2011, 09:57 AM
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Default Re: No Amplification -- Primer Dimer

I agree Sir. I'm worried that my work is lagging. I'm working in Z. mobilis genomic DNA which is the template source too. The product size is 1.6kbp. Components are as usual, the dNTP's, reverse and forward primer, template, buffer and Taq. The trouble is that all I get is a primer dimer. But amplification was fine for 16S rRNA and got a 1.6kb. I checked in NCBI and for Z. mobilis the size is the same. Is there any other way to check my template's purity?
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  #18  
Old 09-22-2011, 06:53 PM
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Default Re: No Amplification -- Primer Dimer

Quote:
Originally Posted by saranyah View Post
I agree Sir. I'm worried that my work is lagging. I'm working in Z. mobilis genomic DNA which is the template source too. The product size is 1.6kbp. Components are as usual, the dNTP's, reverse and forward primer, template, buffer and Taq. The trouble is that all I get is a primer dimer. But amplification was fine for 16S rRNA and got a 1.6kb. I checked in NCBI and for Z. mobilis the size is the same. Is there any other way to check my template's purity?

If you are getting 1.6KB PCR product for 16srRNA, one can assume atleat your pcr protocol was working.

Did you checked for Primer properties Like self , Hair pin formation etc ..

Use this Programme to check your primer properties. There may be problem with your primer design.
[Only registered users see links. ]
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Old 09-26-2011, 06:17 AM
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Default Re: No Amplification -- Primer Dimer

Sir,

It forms hairpin as per the software results. Is there any parameter to zip off the loop? If the Tm is increased will there be less chance of hairpin formation?
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Old 09-26-2011, 07:54 AM
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Default Re: No Amplification -- Primer Dimer

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Originally Posted by saranyah View Post
Sir,

It forms hairpin as per the software results. Is there any parameter to zip off the loop? If the Tm is increased will there be less chance of hairpin formation?
That you have to optimism by gradient PCR
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