ad 1) Yes, this could be. Try to re-purify your samples, also try different purification methods (precipitation, columns).

ad 2) Yes, this can be, although the Tm is unexpectedly high for primer-dimers. Have you run a gel?

Yes, I have run a gel. I ran as an extra control a gel which ran on a normal PCR. Both unknowns gave the target DNA, but the negative control gives nothing like it should be. I have a gel result from a while back from a qPCR product and then the negative control gives a vague band where the target DNA should be.

Do you have a gel from the negative controls showing melting peaks at 86°C? PCRs run under different conditions (you mentioned a "normal" PCR) are useless. Older PCRs are useless as well, since the contamination may have happened lately or got more severe in the meantime (having a faint specific band in the NTCs is an alarming signal!) .

Re-do the experiment. Take the plate and go to a post-PCR-analysis lab (not the place where you pipet new PCRs!) and run a gel from *these* PCRs. I would guess that you suffer a contamination problem...

Hi Jochen, I have got a similar situation with Bas where there was amplification in the NTC. The NTC was in duplicate with amplification in one of the duplicate. We repeated the experiment and the same result again. Could it be primer dimer or what.

If there was a question then I must have missed it.

in my opinion if you have amplification in NT sample

you have DNA contamination in the water that you use

be sure the water is sterilized and use it few times after opening

change the water and you will see the difference

Did you use RNA/DNAase free water to exclude the contamination. Contamination is most likely the cause for what you mentioned. Good luck

Hi

no i did not

normally I use sterilized distilled water

but when the bottle opened i use it maximum 3 times

be sure also to use tips with filter for the micropipette thimes at used for DNA

sometimes the pipette is responsible for the DNA contamination

good luck

@Issa..we used RNA/DNAase free water. @Taha...we used tips with filters.

It kinda funny that there is amplification in the NT sample

@Issa and Taha...will re-autoclaving an alreading used RNA/DNAase free water free it from contamination?

Change the water is better use water not used before

If water is DNA contaminated the re-autoclave will do nothing with it

Do not autoclave! It does not decontaminate nucelic acids. It is rather an additional source of possible contaminations.

If you suspect contaminations in chemicals/water: throw them away, clean the lab thoroughly (EtOH, Isoprop, soap, hypochlorite, ...) and buy new chemicals. Aliquot them somewhere else and do not re-use opened aliquots.

Better change everything (mastermix, primers, water, Mg-solution, whatever you use) together. When you got rid of the contaminations then you may test which of the old chemicals you can further use (one by one).

It could be possible that the contamination is in the tap water. I'm going to try and sterlize it myself first with water in a beakerglass and a walkable UV-Lamp on top of it. Thanks for all the ideas already :)

Note that sterilization is not decontamination. The UV light must really effectively damage the DNA. If the damage is not large enough you may still have a lot of amplifiable sequences. Good luck.

Thank you all

Did you use any specialized solution for DNA extraction?

In my experience, the amplification curves in question are a result of mistaken fluorescence-reading by the thermal cycler in measuring SYBR Green activity; this can be caused by precipitation solutions and/or specialized DNA extraction solutions (Chelex 100, TZ, etc.).

To avoid this, if it is anything related to your situation, I centrifuge my DNA extract so the thick solution is collected at the bottom of the tube and the DNA can be transferred to a fresh tube. If the solutions collected at the bottom of the tube are transferred to the PCR in excess (1), false fluorescence can be exhibited via PCR in real-time.

If your positive and negative control melting curves are similar or identical in peak, you cannot confidently conclude your results and it is likely DNA is present in your negative sample.

(1): Only a small amount is needed for this amplification curve to be expressed.

I am researching Agrobacterium Rhizogenes, a Ri-plasmid, wich is in a bacteria and casues the crazy roots disease. The roots in question DNA is isolated with an universal kit used for several types of compound like soil, leaves, water etc. When I have the pure DNA I make a mastermix with Syber Green and run it on the RT-PCR.

Michael, I didn't understand what you meant so with this new information could you elaborate what you meant with the false fluorescence?

Of course, Bas. I can tell you that I have seen amplification curves identical to your blue (negative sample) amplification plots, however, only when using SYBR green intercalating master mix.

The thermal cycler, as I'm sure you are aware, is shining a light into the reaction tube to measure fluorescence levels in real-time. For reasons I am still not completely sure, SYBR green is capable of producing a false fluorescence level that can be registered by the real-time PCR system as amplification. Personally, I have seen this phenomenon when amplifying Pseudomonas fluorescens (a naturally fluorescent bacterial strain) DNA template, when using Chelex 100 (Bio-Rad), as well as using DNA-ethanol precipitation kits.

SYBR green dye is somehow able to fluoresce in the presence of excess DNA extraction-aid reagent. With the same amplification plot-trend present in Pseudomonas fluorescens DNA amplification, I can say confidently that the thermal cycler is registering some kind of false-fluorescence.

In all I was able to avoid this amplification plot by meticulously separating DNA template from DNA extraction solutions (via centrifuge and transfer of supernatant to fresh tubes) as well as amplify sample in duplicate or triplicate assay for higher statistical chance of avoiding the issue.