PCR unspecific amplification - (May/29/2007 )

Hello, I´m Aline Fagundes and I work with PCR in biopsies of leishmaniasis lesions. My PCR was working well until 2 months ago, when all samples tested (except the negative and positive controls) begin to show several bands of long, unspecific products....I changed all reactives (DNTPs, TAQ etc) but the bands insiste in appear....
I´m afraid that there is a problem with my thermocycler..........Please, what can I do ?????

Thanks a lot for your attention,

Aline

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What annealing temperatures are you using? Try to increase the annealing temperature of your PCR reaction. Also, dont use too long extention times than your expected length of amplification.

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The positive control doesn't show the unspecific bands? Than the problem is more likely to be found in the probe processing. If it was contamination or a problem with the cycler, the positive control should be affeDcted too, shouldn't it? Did you change anything in the pre-PCR steps?

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How about changing the wells of your PCR? I mean, certain wells might have different temperature settings.

How about MgCl2? And yea, please explain in details the procedure.

I am suspecting your DNA template sample.

Good Luck.

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Hi for all,
We run a PCr with another different sample DNA, previously amplifyed without unspecific bands, and the bands appeared ...So, I think that the problem is with the PCR master mix.
When we took the reactive to make the mix we found that the temp of the stock freezer was -14 unstead of -20 º C. Is it possible that the increasing of the freezer temperature could dammage some of the reactives?
Thanks a lot,
Aline
p.s: see attachment

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The fact that your negative and positive controls don't have the extra bands means the problem is not in your master mix, or your pipettes. What is your protocol for template preparation, because that seems to be the next most likely place for contamination or something.
Also, check your PCR cycle details. I have seen cases where temperatures or times have been changed by another person in the lab, typically because they didn't understand how to save changes to a program correctly.

All the best, and I hope you start getting good PCR again soon.

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Just to get a clear picture - your positive controls are amplified with the same PCR ór are they positive controls only for the gel ??? If you have them in the PCR, Swanny is 100% right, it cannot be the MasterMix, then. I would suspect the template, as you must have a lot of different contaminations to get specific bands like this.

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The positive and negative controls are amplifyed in each PCR reaction....Also, it is included in all PCRs a negative extraction control, i.e, a water sample extracted in the same ways of the samples, and did not have unspecific bands.

In the previous experiments I used samples with different time of storage.....

I will extract a new DNA biopsy sample with two different kits, let´s see what will happen....

Thanks a lot,
Aline

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We extracted two fragments of a biopsy sample, one with the extraction kit used previously and the other with a new one.....Let´s see if there is something wrong with the RNAse or another reagent of the extraction kit....
In fact, the unspecific bands become to appear after we changed the DNA extraction room.....It could be environmental contamination in the DNA extration room?
Thanks a lot,
Aline

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Hi for all,
Here is the result of the extraction of the same biopsy sample with two kits, the old and a new one......
Interestingly, we amplifyed another DNA sample, extracted from blood for a different project and ....Here are the unspecif bands again!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!
How can two samples extracted from different materials, with different kits, present the same pattern of bands??????

Please, HELLLLLP`!!!!!!!!!

Thanks a lot,

Aline

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