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pcr smear

pcr smear - PCR - Polymerase Chain Reaction Forum

pcr smear - 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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  #21  
Old 02-24-2012, 04:57 AM
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Default Re: pcr smear

Hi all...M currently working on Sunflower genes. I am encountering a big problem of DNA smear and in-consistent bands since last 4 months in my Nested PCR. I have tried everything mentioned above but no improvement, though i havent tried with diluting or making fresh DNA samples. This is because the same DNA samples give tremendously good results with other gene and primers. My forward primer Tm is 63.0 and Reverse primer Tm is 65.0, I have tried with gradient of 55 to 60 degrees, but no amplification. should i go lil higher on annealing temp?
I have exceeded my project dead-line...kindly suggest.
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  #22  
Old 02-24-2012, 05:30 AM
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Default Re: pcr smear

hello
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  #23  
Old 04-26-2012, 03:44 AM
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Default Re: pcr smear

Quote:
Originally Posted by saly View Post
smear may be means som hydrolysis


I do not understand what you said about
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  #24  
Old 04-26-2012, 03:45 AM
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Default Re: pcr smear

Hi,

Thanks very much for this comment. It help me to think about my ideals.

Tks again and pls keep posting.
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  #25  
Old 07-19-2012, 08:11 AM
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Default Re: pcr smear

Try to get the salt concentration optimised for your primers.
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  #26  
Old 01-11-2013, 07:47 PM
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Default バーバリーシャツ

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  #27  
Old 01-15-2013, 08:54 AM
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Default Re: pcr smear

Quote:
Originally Posted by bhaktamehul View Post
Hi,
i want to know what are the causes of pcr smear. coz i have been having some smear from last one week , but early it workd great... i even tried new dna samples, made changes in annealing temp and time and also used same primer from other lab(where it is working),,

so i just want to know what can be the causes of smearing , so that i can sort out problem...

mehul
Hi
The first reason for smear is DNA which was degradation , must prepare new DNA fresh with good concentration and try again .
cheak the primer on the agarose gel
good luck
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  #28  
Old 01-15-2013, 08:57 AM
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Default Re: pcr smear

Quote:
Originally Posted by pooja pandya View Post
Hi all...M currently working on Sunflower genes. I am encountering a big problem of DNA smear and in-consistent bands since last 4 months in my Nested PCR. I have tried everything mentioned above but no improvement, though i havent tried with diluting or making fresh DNA samples. This is because the same DNA samples give tremendously good results with other gene and primers. My forward primer Tm is 63.0 and Reverse primer Tm is 65.0, I have tried with gradient of 55 to 60 degrees, but no amplification. should i go lil higher on annealing temp?
I have exceeded my project dead-line...kindly suggest.
Hi
The first reason for smear is DNA which was degradation , must prepare new DNA fresh with good concentration and try again .
cheak the primer on the agarose gel
good luck
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