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Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )

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Thanks for the answer, I'll definitly try it.
What is another mistery on this smear problem, is that the smear comes all the way from the well itself (it means they are big fragments). Isn't that strange, considering that my taq polymerase limit of amplification is 1-1.5 kb?
Thanks for the comments to this and my other question.


Sorry the ignorance, but what the DMSO would do in the PCR reaction?


you can try 1-10% DMSO concentration. But most optimal result is obtained in 3-5% DMSO. At 10% or beyond that amplication reaction usually failed.
Best wishes

QUOTE (Pablo @ Nov 7 2005, 04:27 AM)
Sorry the ignorance, but what the DMSO would do in the PCR reaction?

-Amit Kumar-

The DMSO worked well, thanks guys. Is not like when the samples were fresh but it's not too bad neither. 8% was the best concentration for me actually. Have anybody tried Betaine? do you guys think it would improve it?


QUOTE (PCR-extension @ Oct 4 2005, 03:23 AM)
Does anybody ever doubt the primer itself? I have had a PCR problem before. It turned out that the company gave me the wrong primer. When I resynthesised the prime, promlem solved. mad.gif

Oh I would be so upset with the company. I already get very upset when I do a mistake.... if that would happen to me Grrrrr mad.gif


QUOTE (Pablo @ Nov 6 2005, 11:57 PM)
Sorry the ignorance, but what the DMSO would do in the PCR reaction?

DMSO and glycerol are adjuvants in PCR reactions ; they improve amplification efficiency and specificity of PCR, when used in concentrations varying between 5-10% (v/v).
Because of contraditory results the usefulness of these adjuvants needs to be tested in each case.
DMSO is a solvent that helps relaxing DNA just like a detergent can do.
(Please Correct me, if Iam wrong!)


keeping in mind, that u have a faint desired product with a bright smear....i suggest just do a phenol chloroform extraction, precipitate with ethanol over night and use it as template.........and well repeat everything afresh.........not being lazy lets see it will work................................


well i also feel DMSO is used as it helps in the denaturation of template DNA especially you have a long target.......i remember i used it once to amplify my 1.6kb too missed out the pcr product initially once i started adding DMSO it was fine and good.........sometimes there is no complete denaturation of the template which is crucial for succesfull pcr amplification.....



First check ur DNA is correct ( Not sheared )

then check ur primer for Annealing temp

Temp of Annealing = +/- 5 Temp of melting

Temp of melting= 3 (G+C) + 2 (A+T )

try this

Best of luck

Bye wink.gif


QUOTE (kudna @ Sep 22 2004, 08:24 PM)
Thank you for your replies. It turns out the carry-over contamination may infact have been the problem. After making up fresh solutions and ordering new primers, taq, dNTPS's etc, using a new set of pipets, and setting up post-PCR in a different lab (don't know which of these or how many of these solved the problem) the streaking has disappeared and the bands are bright.
The Mg2+ concentration I'm using is 1.6mM which I haven't changed so I don't think that affected it but thank you for the suggestion, and the thank you for the troubleshooting site recommendations Ihab. Very helpful.


Yes, but if smearing occurs again, it's possible due to working with chromocomal DNA or using too much template. Smearing can also happen by using plasmid DNA as a template if it happens to be dirty. Be sure to change tips at every pipeting and not have them placed behind the trash tip container so that possible aerosol or even small drops drop into your tip holder. Good luck! smile.gif


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