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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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non specific pcr products
Posted by: Kirankumar (IP Hidden, Unregistered user, )
Date: April 16, 2005 02:40AM
I had standardized PCR for 530 bp. but later in same pcr conditions i am getting nonspecific bands along with my 530 bp product. i tried temperature gradient taking all new aliquotes of pcr ingredients. what should i do now?
Re: non specific pcr products
Posted by: P103 (IP Hidden, Unregistered user, )
Date: April 16, 2005 03:44AM
1.Did you increase the annealling temperature. In my case, I increase as a series (T+3 C).
2.Try to purify your PCR product at 530 bp from agarose gel and amplify again. 3.Check the length of primer. good luck
Re: non specific pcr products -- me too
Posted by: forest (IP Hidden, New member, 1)
Date: April 21, 2005 08:17PM
I am also facing similar problem. The optimized annealing temperature still gives me some light smearing (or sometimes faint non-specific bands) background, but with the desired band clear.
And most striking is, after I did gel purification (cut out the desired band for purification), I still have few more non-specific bands (with bigger sized of desired band). Could some one please help me?? Could it be something to do with my template DNA for PCR? I found that only the newly extracted DNA samples gave me this problem. The old DNA samples were doing quite well (appeared to be a single band after PCR) with the same annealing temperature, parameter and primer-set.
Re: non specific pcr products
Posted by: l.anbu rajan (IP Hidden, Unregistered user, )
Date: April 27, 2005 01:52AM
sir
iam l.anburajan from india. iam doing my research in identification and characterization of osmotolerant bacteria. one of the gene included in my study is ectoine. while pcr ampli. iam getting non specific products in this gene. its tm value is 47. i have tried with various annealing temp. ranging from 45-52, and i have reduced the template conc. the result is negative (i.e) iam getting extra bands along with the desired pdt. i humbly requesty you to kindly consider my request and give me your valuable suggestions. thanking you
detection of transgene by pcr
Posted by: pcrgirl (IP Hidden, Unregistered user, )
Date: April 28, 2005 07:03AM
I am doing a pcr on a certain transgenic crop but my problem is that i'm getting nonspecific products. And my expected amplicon is very very faint. I already did a touchdown. I also increased the DNA in the reaction (100ng). And i think the components of my pcr cocktail is ok coz i'm getting a very strong band in my plasmid(as my postive control). I want to have a distinct and strong band to validate the presence of the transgene in the crop im working on, could you please help me? Thanks in advance.
Re: detection of transgene by pcr
Posted by: devanarayanan (IP Hidden, Unregistered user, )
Date: April 30, 2005 11:33AM
hai,
Have you checked the amplification using nontransformed plant DNA with the same primers.
Re: non specific pcr products
Posted by: abdel (IP Hidden, Unregistered user, )
Date: May 25, 2005 03:28PM
guys
you have always the option of gel exracting the band of your interest,why do you care about the other band (non specific bands) good luck
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