I used PCR for amplification of COI gene of Sparidae fish , 1st the samples shows good quality bands at 55oc annealing temperature.
but after 3 months I do the same reaction for new samples of the same species, but the bands show smears !
I have used agarose gel 1.4% with TAE buffer, 200 volt.
Hi, I think that perhaps it might be because of the DNA. Perhaps, when the DNA samples were recent they amplified ok, but then because of contaminations in the DNA it could have started to degrade generating problems with the amplification. Perhaps you can check if the quality of the DNA is ok and if it is still ok after storaging?
It is an idea, perhaps it can help you. :)
Thanks Diana
Is there a method to check the quality of the DNA other than check the amplified gene by gel electrophoresis after PCR ?
Well you can check your DNA visually in an agarose gel, observing that it consists in a defined band without smearing... Other alternative is to check absorbance in as spectrophotometer or a Nanodrop equipment. DNA absorbs at 260 nm, you can check 260/280 and 260/230 ratios to stimate DNA purity.
Amit , Diana thanks alot
I have checked the DNA before in an agarose gel by 2ul template and 2 ul dye but no bands appeared although i had one sample of them as a control (which already had band after amplification to the gene ), what may be the cause ?
DNA is probably degraded.. was it stored at -20? Nanodrop and find the conc. Primers may have been stored improperly, the PCR mix may have been at 4deg for long
(hence looses its amplification efficiency).
Do you amplify GAPDH/endogenous control along with your gene of interest?
Good Luck
Hi, recently I am also experience the same thing as you mentioned above. Could you tell me how you solve it?
Thanks
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