Everything before is right, primer dimer is classic case of amplification in negative controle, but it normally appears late in cycle number, and the melting temperature is different from target amplicon. Moreover you can check the size of the amplified product on agarose gel to see if it correspond or not to amplicon size.

On the other hand when you perform the PCR of the same target a lot of time, for long period, you should have aerosol of the target product in the PCR room. So as suggested by Anna, it is important to have different room for PCR preparation, run, and analyse. If your contamination persist, just try to prepare your PCR reaction in a never tried room, even in an office!!!

Good luck!

There could be a lot of different reasons why you still have contamination in your samples. What is you target? For instance, if it is the bacterial 16S rDNA gene, we have some experience of contaminated reagents (water-living bacteria mostly), but it has not been in every Lot for the kits we used.

When did the contamination first show? From the first run or after some runs?

How is the lab set-up? Do you perform pre-PCR and post-PCR work in the same room or working area? Do you use gloves and change them often? Use DNAway or bleach or similar agents for cleaning up DNA contamination? And so on... I wish you best of luck!

Some times due to aerosol (DNA in the air) it happens, so the best thing to do is do the PCR set up in a seperate place, use the DNA in a seperate station. Each part needs to be done in a seperatestation, or place that might help the contamination.

Thanks everybody for your answers. I am not running negative control, nor positive control. My reactions just contain SYBR, primer and water and I use clean gloves, mask, lab coat, and protection for my hair. My reactions have been contaminated since last year, and I had changed all reagents. These curves looks weird, so it can be primer dimer. I will check my PCR stuff again using tested primers to make sure that it is not a primer dimer case. Thanks again ;-)

I think you got answer, its due to contamination of template, so u just replace one by one and check wt is adjactly contaminated with ur template dna...

Everything before is right, primer dimer is classic case of amplification in negative controle, but it normally appears late in cycle number, and the melting temperature is different from target amplicon. Moreover you can check the size of the amplified product on agarose gel to see if it correspond or not to amplicon size.

On the other hand when you perform the PCR of the same target a lot of time, for long period, you should have aerosol of the target product in the PCR room. So as suggested by Anna, it is important to have different room for PCR preparation, run, and analyse. If your contamination persist, just try to prepare your PCR reaction in a never tried room, even in an office!!!

Good luck!

You should run a gel of the qpcr products to confirm if its contamination or just primer dimer.. Sometimes primer dimer may give large products as well. In this case you need to change your pcr conditions. do you see the same peak (in dissociation curve) in your test samples also as it is in the negative control ? and what is the Ct value of your contaminated sample? If its higher then its not a contamination

Did I understand you right: after changing the qPCR master mix system from Qiagen to Applied Biosystems you have these amplifications in no template controls? From my own experience I must say, that the Applied Biosystems qPCR master mixes seem to be less stringent than Qiagens -> it´s much easier to get spurious amplification; especially in the -RT negative controls and sometimes even in the NTC (no template controls). So you either switch back to Qiagen and check if your problem persists or you design new primers from scratch and test those.

Also compare melt curves of your putative NTC products with those of samples that contained template. Are the peaks the same? Primer dimers usually have funny looking peaks of lower melting temperatures than "real" products. If you need more help, post screenshots of amplification and melt curves of samples, NTC and -RT-controls...this will help interpretation a lot :-)

Another thing I´d like to know: do you have this problem with all of your primers or just one set? From which organism will your templates be from? If bacterial, primate/human, I could see contamination problems - otherwise, don´t get too paranoid.

Also, if "contamination" comes more than 10 cycles after your sample, the degree of contamination is so little, that it will contribute only 0.01% to your sample´s signal - thats orders of magnitude less than the deviation you have between biological replicates...

Thanks Christoph for your reply. And yes, the contamination issues had started around the time I had change the master mix brand. I had the same problem with all my primers. I changed all reagents including all primers set. Same thing....

Previous answers told me about primer dimer, so I repeated the PCR running using a primer set which I had tested previously and it worked fine. Unfortunately, more amplifications curves without template....

Do you think that new sequence of a working primer sequence can generate primer dimer? I did not run gels yet, I will run these samples on Monday, but I have PCR computer interface. They are attached.

Melting curve of one of my targets....

Your melting curve dont look fine. Firstly, its difficult to differentiate the NTC with your target in this picture as its not labelled. They have a big shoulder before the actual peak and also few shoulders (which could be neglected) beyond your peak. So your PCR reaction conditions might not be significant. May be increase the annealing temp or decrease the extension time to avoid such issues.

I have heard that many of the qPCR kits have had problems with contaminating DNA in the kit. I've heard that the Promega qPCR kits tend to be clean - but I think they're probe based.

Shrey, these PCR reactions are triplicate: SYBR, water, primer. No template....

I will run a gel with these products and run another PCR following your recomendation. Thanks for your reply. I really apreciate.

Your melting curve look strange. The pic is large and not clean, and moreover the Tm is low. It could primer dimers, with different kind of dimers.

Again, try to prepare your PCR in another room, even if it seems creazy to you, it doesn't take you more time and it could change your result.

Another point, have somebody in your lab ever tested the master mix you use with the thermocyler you use?? You have to know that some mastermix are not compatible with some machine. For example the Promega master mix which is quite good with a lot of machine doesn't work with the machine from Roche (LightCycler).

You should keep a positive control (containing a test cDNA) and you NTC to compare.. These peaks can be of dimers and to be sure you neeed a cDNA.. you can first prepare your mastermix with primers and sybr green and then pipette it first and close the NTC tubes/wells. Then take out your cDNA and add to your reaction so that you avoid any contamination. It would be good if you pipette your NTC in a separate place to avoid any other spurious dna getting into your tubes. Still if you get similar peaks, compare the threshold values in the melting curve. The dimers usually have a low thershold than the actual product and also they appear earlier in the melting curve.

Richard, people from my lab work with the same kit I do, but different templates. Do you know why the Promega's kit doesn't work with Roche PCR??? It is unusual....

Thanks guys. It was very heplful.

No idea Aletheia, we tried it and it do not work, and the Promega staff answer us that we got the same result as previous lab which have tested their kit...

But is seems to be not your case if collegues from your lab use the same kit successfully. My idea is still aerosol for you. Please try to prepare in an unusual room your PCR.

Hi Souzia,

PCR is the nightmare if it does not work!!!

i would design my control in a way so that i do not waste my time trying to optimise different reagent or condition.

I would at first place do the RT synthesis with all controls

1A---All RT reagents without your RNA (this serves at negative control to test your RT reagents for contamination)

1B---All RT reagents except RT enzyme + RNA (this serves at NO RT enzyme control to check for DNA contamination in your RNA preperation

1C--All RT reageants + RNA (Test sample or cDNA)

Then while setting up your PCR you should include

2A---All PCR reagents - Primers - DNA (this is negative control for your PCR reagents)

2B---All PCR reagents + Primers (This along with 2A will be a negative control for your primer contamination or any primer dimer if your primers self complimentary)

2C---All PCR reagents + Primers + DNA (this is your test or positive control)

here 2C can be done with 1A, 1B and 1C if you suspect contamination from your RT step.

Now looking at the amplification plot with your no DNA template reaction, it looks like the primer dimer which are strongly self complimentary. Try to check the 3' and 5' self complimentarity for your primer. This is the most probable reason for your primer dimers. Below are some suggestion for designing good primer. I normally do these checks, even if i use primer designing softwares.

Try to make the primer with rich in AT at 3' end, A and T are lower possibility of mispriming than G and C. At the same time try to end your primers with C or preferably A as these bases do not wobble, and primers designed will be more specific.

Very important of all is not to design primers with 3' self-complementarity. If you cannot avoid 3' self complimentarity (which some times can happen when differentiation isoform), then you have to be 100% sure to avoid 5' self complimentarity. Primers ending with GT at 3' end are not usually good because of wobble effect that can make them self complimentary.

If self complimentarity is not the issue, then it could also be possible that your primers are contaminated which can be checked by running a PCR reaction as mentioned in 2A, 2B and 2C.

i hope this helps

Contamination or primer dimers

Very much looks like primer dimers. These can occur if you your initial primer concentration is too high, if there is no template to be amplified or if the primer design is bad. Try using 25% or 50% of the current primer concentration and try a new set of primers.

Thanks Izhar and Christoph for your explanations. For primer design I used Primer3Plus and OligoAnalyzer, which I use to check primer parameters, such as self dimer or hairpin. I ran an agarose gel 2%, and it is a primer case (fortunately!).

Agarose gel 2%. Primer dimer....

Most likely you have a contamination issue. It could also be primer-primer amplification. Try just running a standard PCR and then run it out on a gel. Try all combinations of primers from no template to single primers, every combo you can think of. That will help figure out what the issue is.

is the gel shown above No template PCR? If yes then i would think this to be related to higher concentration of primers used.

Since you have mentioned using 25nmol of primer. If i understand it correctly, and suppose you do a 25ul reaction this would correspond to 1mM of final conc., which is very high.

I would tend to use primers at final conc. of 200-400nM.

No Izahar, my final concentration of primer is 0.5uM in a 20ul reaction. But I am concerned about my master mix. I never had this kind of problem with Qiagen master mix...

I think reducing primer conc. or increasing the annealing temperature might help to increase the specificity of your reaction. Try a run 1 degree higher.. your melting curves should also look good with distinct peak (of primer-dimers if any and template) and shoulders.

An alternative, though more expensive, is to try hybridization probes (TaqMan, Scorpion) etc., thereby preventing the detection of the primer dimers.

We have had the same issue with primers from Life Technologies, try reordering the primers from IDT.

Everything has been covered except for one thing - your gloves. Do you spray your gloves with 70% ethanol before setting up your master mix? I previously had contamination issues that came down to gloves. Most gloves have a thin powder covering and this can wreak havoc with any sort of PCR if the powder inadvertently gets into your reaction. I always spray my gloves down with 70% EtOH before spraying down my bench and pipettes and letting them dry. I also spray down my gloves after using the vortexers/centrifuge etc etc. Just a thought.

Hi Eileen, I spray ethanol on mmy gloves. And I also spray on my bench, pipetes, and I use UV light for 5min at least....Thanks for your reply ;-)

Actually, I don't know. We always use 70% ethanol... Why methanol would be better?

Thanks Bastian. How about Isopropanol?

Just check whether your primers form extendible pairs or not, because you may be getting false positive results just from the extendible primer pairs. I would suggest you try both primer blast and oligo analyzer of IDT to check the same. Good luck

REAGENTS!!! might be the culprit in ur case. U need to check with -1 reagent each time ( use from one of ur trusted friend or order some if u could) and troubleshoot which one is contaminated. BTW, what are ur samples and how did u isolate the nucleic acids? Even ur water from Ambion could be contaminated. Fingers crossed but u need to chk one by one.

Use of 0.2M HCL can be a simple alternative since it can de-purinate the pre existing amplicon.

We do lots of Universal PCRs. One thing we do in our lab is we UV both our mastermix and primers for about 15 mins in the clean spot. In our lab, we have come across contamination from E. coli, for the Taq polymerase (in the master mix ) is probably in recombinant E. coli. and when we talked to the manufacturers, they told us that they did not guarantee the the Taq they provided did not contain DNA. But usually, under very stringent condition we go about UVing everything possible. And yes, that do not affect the amplification process.

Aletheia,

Introduction:

If it is primer dimer the half concentration of primers should decrease the amount 4 fold, that is the CT should be increased by 2.

I saw the amplification curves and the gels. What are those curves? All negative controls (no cDNA)? The melting curves are in concert with my observation that if you have good reagents and you do not give the polymerase any template to work on, it will just find something for itself.

Results and Discussion:

My suggestion is like a Gordian Knot. Do not worry about this whole thing. As soon as you add a healthy template, this reaction that the polymerase struggled to carry out will disappear, and you will not see any trace of it neither in the melting curve, nor in the gel that you run with your nice amplified product.

Already your erroneous CT is about 31. If you reduce the primer concentration, it will go higher, I bet. Even if not, think about this one:

If you add template, make sure that the template is in a concentration that is high enough to result in much lower CT than the CT without template.

Now, even if you do not believe me that the false reaction disappears, and you say that the "contamination" is still there and it in itself gives a CT31-generating amount, and you have -lets say- CT24, then the sum of the two templates give you 2**(31-24) times more amplicons than the negative control used to. This is 128 fold difference, that means less than 1% of the amplicon is "error", more than 99% is "success".

If you can make 10 CT difference, the putative error will be less than 0,1%. None of your pipetings and reactions can be as accurate as that.

Conclusion:

You are doing everything fine. Do not worry about reagents and gloves and stuff. (You may titrate the effect of the dilution of the primers on the positive (cDNA added) reaction or the effect of slight increase in annealing temperature on both the positive and negative reactions, though.)

Enough template will solve the problem. Watch out for CT values. And gels and melting curves of the positive amplicons always.

Thanks Peter for the amazing explanation. Actually, I repeated my reaction using template, and they worked beautifully. I ran another gel, and it was primer dimer for sure. I am resistant to change the primer concentration because I am in the middle of my project, and 0.5uM primer /reaction worked in the beginning, so I was going crazy because my protocol was working and after while it stopped to work. After discuss the findings with a lot of people, the misterywas solved, and my amplifications curves on my blank reaction was caused by master mix, or because I changed the brand. Thank you everybody that stopped by to help me. I really appreciate that ;-)

PS: I attached my last PCR (temperature gradient).

Hi Aletheia,

I am in the same trouble as you were and all my NTCs had amplification,could you tell me how you solve this problem, thank you!

Hi Aletheia,

You have to test one by one all your reagents if they are contaminated or not. Also sometimes aerosol in the room or working bench gives false CT values and  melting curves, so work in a very clean area inside a bio-safety hood and see the results.

Hello Erika,

I am facing the same problem as you are. Have you been able to figure out what was going on with your PCR? 

Working in laminar flow and use all unopend reagent including water. Prepare new stock of primer.