Attached is my file from a Biorad CFX96 Real Time system. The red lines are standard curves, the blue curves are monsters with different concentrations and the green curve is a negative control.
I know that negative controls that come up after cq 30 are unreliable. But my question is the weird blue curve that rises much more slowly then the other curves. I know for certain that the target DNA is present in the unkowns. Could the matrix in the unkown be an interfering factor? That could explain why my lower concentration of unknown doesn't even come up even though my negative control does come up.
A 2nd question, can a primer dimer have the same meltcurve as the target DNA? Because that could explain the negative control as I can't think of anything that is contaminated as I have replaced each component for a clean alternative.
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.
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.
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?
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