Troubleshoot PCR smear problem - all I got was smear!! (Aug/18/2004 )
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.
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?
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
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 target.......me 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 )
Best of luck
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!