contamination in real-time PCR - (Apr/01/2005 )
I also had 'positive' negative controls.
But only when using universal primers for prokaryotes. (also sometines with other, but than over 40 cylci)
And ... when using E.coli specific primers.
Talked with some guys from a company here who also use real-time PCR.
They overcome by NOT using kits with SYBR Green and dNTP's mixed and the expose their Taq 3-5 min to UV-light.
It's known from literature that kits often contain DNA from the organism the taq comes from. That's often E.coli.
I'am new here, so maybe I respond on 'dead' topics, but I'll try to find my way.
I'm now trying a new real-time machine and got strange NTC "contamination" with mouse beta-actin primers in SYBR kit.
Usualy comes late in the run, but what is interesting, melting temperature is significantly lower, than that of the specific product. It's in the area where I would expect primer dimers, but I don't see this peak in actual samples.
What is strange, that it doesn't happen in all NTCs, only in one of three or two of four (all from the same master mix, only the water-as-sample pipetted separately).
Only thing I was thinking about is that the amplified NTCs were always in the border of the wells on a plate and that some temperature differences could cause the primer dimer formation. But this theory seems a bit far-fetched. And the machine (LC480) is supposed to have a novel class block without any edge effects or so.
Hi kpmitton,
Thanks for the useful advice you've given concerning contamination in QPCR.
I am using the SYBR green mix from Thermo Scientific and my controls are showing amplification at the 39th cycle and above. The melting curve shows a peak for the controls though its not as high as that of my samples. the Ct values for my smaples are in the 20s.
Should I ignore this or is it something to worry about. Is it due to primer dimers or contamination?
Thanks,
1) you must sequence a band from your reaction (gel extract or pcr cleanup), to know what you are looking at for sure..(if the band is not that different in size from your intended amplicon.
2) reactions without a template will often, more often than not, give a band eventually. Without template, the even the most inefficient primer interactions will eventually make a product (on their own). While they may not be efficient at first, eventually the first primer dimer products will become very efficient template for primer dimer product formation. So a bona-fide negative sample can give you these bands.
3) As a rule of thumb, you can assume that primer dimer bands, or even mild true contamination, that is MORE than 5 Ct. values away from your samples/standards are ok to use. 10 Ct. would be better.
4) Make sure you start with a clean area, and try to set up your PCR away from where PCR reactions are opened (ie where gels are loaded!)
5) you can UV irradiate your tubes (open) and even your mastermix before you add dye and Taq to destroy TEMPLATE (thymidine dimers formed). this usually does not seem to harm primers or dNTPs in the mix. irradiate (5 min) your pipettors too, facing up in a beaker. Use barrier filter tips.
6) DO NOT take any template out of storage untill you have made yoru mastermixes then put all the master mix components back into storage.
7) do not be afraid to run real time reactions for far less cycles (ie. not 40), such as 25-28 cycles, if your samples are showing product build up by that time. the melt curves done at that time, and checking the reactions on a gel will show if your product is popping up long before primer dimers or mild contamination do.
good luck.
Ken Mitton, PhD
Assistant Professor of Biomedical Sciences
Oakland University Eye Research Institute
http://ken.mitton.com
Ignore this. 39 cycles is well past the point where anything can happen. It's 19 cycles past your sample values, or about half a million times less signal.
Hi,
From my experience i can tell you that it is due to primer dimer. I was also getting signal in Blank but when i change the primer problem is automatically solved. Using nuclease free water from different company......will probably not work.
Good luck
Prabhakar
Hi,
i thought primer dimers shd appear as a small bump or peak ealier than the true amplicon in the melt curve? shouldnt i also see this 'bump' in my samples? am only seeing it in the control no template. when i run the samples on agarose, the control does not show any bands? what does this mean? some times i get Ct values lower than or very near my samples, but no band on gel? shd i trust my results. i change my water every day, use autoclaved tips and also started autoclaving the pipetts. am frustrated and not sure what to try next!
Hi, I want to ask a question about polypropylene PCR tubes. Before I perfome real-time PCR, I put polypropylene PCR tube package on the flow laminar cabinette under UV for 30 minute. Does UV radiated polypropylene PCR tube effect real-time PCR reaction? In addition, polypropylene tubes were advised that mustn`t use for transfection with DNA/lipid complexes in some sources. But there use polypropylene microcentrifuge tubes in many commercial transfection reagent procedures. Have you ever experienced for DNA/lipid transfection with polypropylene tubes? And did you put tubes under UV before real-time PCR or transfection? Is there any harmful effects?
Best regards
Best regards
In addition, there were reported that polypropylene tubes are poor resistant against UV in some resources. How does this poor resistancy effect master mix (master mixture prepare in polypropylen tube) and real-time PCR reaction?
If you still get contamination although working "contamination" free try to solve your problem with AmpErase (UNG) using a dUTP containing master.
AND NEVER no NEVER open a PCR rxn after cycling in your prep lab, or pipetting preps and amplified samples with the same pipett.
Sometimes it also helps to wash your labcoat from time-to-time 
Have you looked into your master mix concentrations? MgCl2 concentrations being too high or too low can create smears of non-specific binding. For 100ng/ul DNA concentration I use 1mM MgCl2. I go to 1.5mM for certain sequences as well.