Result! I ran the reactions again with separate areas/pipettes for set up, addition of template and analysis as suggested and got good strong positive control bands and NOTHING in the NTCs. Guess that's what I'll do from now on! Thanks all

Hi, are you working under a clean bench? Did you check your Eppis? Cheers, Nadine

Hi Nadine

I've not done the reactions in a hood etc, just on the bench as normal, although cleaned everything before working. I've performed the reactions in both sterile 96-well plates (real-time format) and in standard PCR tubes and get the same in both cases...

D

David, have you used new probes? Do you work on ice?

Khalid

Hi, After checking all your reagents/ consumables and doing the test by another user it may be that the contamination is happening in the gel! Are you not doing real-time PCR?

Hi David, try to prepare a new 96-well plate only with RNAse-free water and resetting the background reading each fluorophore (I don't know what kind of qPCR machine you are using) you use.

Your lab air is contaminated with product - try setting up the reactions with CLEAN unopened reagents with UV-irradiated pipettes in a hood. This happened to my lab when we were doing long PCRs had to move the lab and make sure that our pre-, PCR, and post-PCR anlaysis were done in SEPARATE ROOMS! We have a thermocyclers in a separate room (where we set-up the reactions in a hood) and never open the PCR tubes after a PCR run in this room.

We have tested decontamination methods and found that EthOH is not sufficient to remove amplicons - even ones left to dry on the bench. You must use a dilute mixture of bleach to destroy them. I would recommend repeating your cleaning procedure with a 10% bleach. Second, double check your gel buffer - it may be contaminated with the amplicon.

Possibility 1

In the vast majority of cases the contamination comes from your pipettes even if you use "barrier" tips or your hands.

All manipulations of master mixes,primers, PCR water and enzyme should be done in a dedicated area with dedicated pipettes. Have a dedicated box of gloves for the above manipulations.

Loading and manipulation of PCR products should also be done with pipettors dedicated solely for that purpose in an area far from where you mix reagents or add template.

Wear gloves when you load gels and work with PCR products

All or some subset of your reagents may be contaminated with PCR product. It may be easiest to just order them fresh and follow the above rules.

Remember that a PCR reaction produces an astounding number copies and they are highly amplify-able and stable.

Possibility 2

You are getting primer dimer that is the same size as your PCR product. This is fairly common. Do melting point analysis on a real time unit. You can try cloning the questionable product and sequencing it...

Perhaps the plates might be contaminated if old?

Another source of contamination could be through human skin so I would recommend wearing gloves and a clean labcoat.

Care must be taken while sealing the plates with the plastic lids as that process can introduce contaminants.

If all fails, I would also check the qPCR instrument. Though unlikely, aerosols etc present in the machine could lead to contamination. Similarly during centrifugation of the plates, contents from one well can stick to the lid and be transferred to another well...so careful handling of the plates is needed.

Perhaps you could keep some wells with only distilled water and check if there is a signal in those wells as well and it would give you an idea. Testing on a different machine might also provide some clues.

Goodluck with the troubleshooting!

David. Bennett nailed it on the head. As an FDA toxicology lab for gene therapy vectors, we have to get the NTCs to "0" on real-time PCR. We are isoloated behind two doors in the basement. Reason being the vector DNA is in the air system of the building after all these years. In addition, DNAway and 10% before ETOH can help significantly. Change your water as well.

Hi David,

First of all, what genes and what organisms are you targeting? Are you using universal primers or specific primers?

All real-time PCR reagents are contaminated with nucleic acid - often from the recombinant bacteria that the enzymes are purified from and sometimes from the water used for production (as well as a number of other sources). This is a big problem when targeting E.coli for example as well as when using universal 16S rRNA primers.

If this is not your problem, then a good idea is to buy new primers and go to a colleagues lab to perform the PCR using their reagents (if you have a colleague willing t let you do this). If you can avoid contamination in a different location - this would point to a problem in your lab with contamination due to a lot of PCR being performed. This would then require a thorough decontamination of everything using DNA and RNA Zap.

I hope this is helpful and good luck with fixing your contamination issue.

Justin.

Thanks for all the contributions!

To respond to a few comments, the problem is almost certainly not primer dimers, since the products in NTC have the same Tm in melt curves (qPCR format) as those in samples with cDNA, and when run on a gel they are specific to the gene assayed/expected product size (ie I am assaying several different genes with specifically designed primers that give different product sizes; these are reflected in the size of the NTC bands).

Also, it cannot be in running buffer as I'm not adding any! The polymerase I use for standard PCR (MyTaq red) comes with a red dye already in.

It seems the consensus is that contamination through pipetting and preparing in the same area can occur quite easily, and so the obvious thing to try (Thomas; Bennett) is separation plus a hood, in addition to using fresh/dedicated stocks of everything (which I've already done!). I will try this and get back to you!

As many mentioned above you may have contamination in your lab. Try working in a separate lab with all new reagent aliquots and see if that works. If so, separate your pre-PCR and post-PCR work areas, and use bleach and/or UV to routinely decontaminate your pre-PCR space.

There is also a possibility that these primers are picking up other products. If your NTCs are truly only water blanks, then it may be primer-dimer or be picking up DNA contamination in your reagents (from the manufacturer), particularly bacterial. That seems unlikely, however, if you're trying multiple different primer pairs. If your NTCs are actually -RT controls, it may be that your primers are amplifying residual DNA which would certainly be variable from run to run.

Although your sample running buffer may not be the source, have you tried fresh buffer used to prepare your gels?

The suggestions to separate the mix preparation and post-PCR processes are excellent points and should be standard for all PCR labs, but do not help once the contamination is already occurred. Unless, however, you can move to an entirely new lab space.

UV sterilization is a great method for mitigating contamination of plastics, but one must use a UV Stratalinker (typically used for crosslinking Southern blots), the UV light in most laminar floor hoods is not direct or intense enough to crosslink amplicons or DNA unless left for very long periods of time.

You are using tips with filter, right?

Air can be a source if you work on bacteria, fungi etc and you work on conserved genes.

As usual, many very good suggestions.

There is one key question you have not answered is: What are you trying to amplify? Taq and other enzymes, even when highly purified, still contain microbial nucleic acids that will amplify.

David: I think its definitely the air, think about it you only need the volume of ONE E.coli 1 femto-Liter to introduce sufficient PCR products ~ 100 copies for a carry over problem. When you open the tubes on your bench you are seeding your PCR reactions with your carry over product.

Set-up your reactions in a hood in which you have left the UV light on for several minutes - UV DNA damage will block amplification of any DNA that might be there.

Good luck, Ben Van Houten

Hi David,

Is it SYBR Green or TaqMan real-time PCR? If your primers and probe are specific the only most likely factor causing this problem is DNA contamination as many other people also suggested. This contamination may occur from your reagents, equipments and your work station. I would suggest to perform your real-time assay in another place and see the results. Hope it may help to find out the source of contamination.

Cheers, Jeyaseelan

Hi David,

You haven't mentioned what are you targeting? Your target is so important, for example if you are looking for something close to murine leukemia viruses you will find TaqMan probes and Master mix contaminated with these viruses already even the columns used to extract the DNA reported to be contaminated with these viruses so whether you take extreme caution to set up and run those assays you would still detect amplification signals in your NTC well. It would be good if you do your due diligence in your lab and see in terms of PCR, what kind of target has been used before, and start a thorough, an extremely thorough, decontamination process of the hoods that set up your master mix, and as Brock says UV radiation always works. UV everything for at least 20 min prior to set up.

Good luck.

Thanks again for the suggestions. My sample is from rat brain, and I am targeting c-fos and vasopressin, as well as 18S and GAPDH as reference genes, so I'm guessing the suggestions re likely bacterial/viral contamination in air/reagents are not relevant here; it seems more likely that I have managed to contaminate my working area with cDNA and therefore the idea of moving to a different area to set up and using a hood would be the best move...I just have to find out where I can do this and then I'm going to give it a try!

As many mentioned above separating the different steps (separate rooms for pre-PCR, DNA handling and post-PCR each with dedicated pipets) should get rid of most sources contamination.

From your answer I take it you are not running real time PCR, so you have no indication about the NTC reaction kinetics or how much amplification there is?

One last possibility is that the contamination is happening while loading the gel. I assume you've tried leaving several empty lanes between your samples and NTC + loading the NTC last?

Good luck.

Result! I ran the reactions again with separate areas/pipettes for set up, addition of template and analysis as suggested and got good strong positive control bands and NOTHING in the NTCs. Guess that's what I'll do from now on! Thanks all

Always separate these area.

Great.

Great news! I am happy that now you have identified the source of contamination.

Hi there,

I strongly agree with many of the above suggestions, as i assume that you have bench work contamination! whether its bacteria or else. You have done good job with the troubleshooting, the only thing you may try to do is to UV light all your stuffs of course except the reagents and move to another area perhaps different lab and try again. Lets hear from you. Best

Great, just saw that you have succeeded ;)

Hello,

my qPCR shows almost no difference between the sample and control, which I could see clear difference via western blot. do you have any idea about what the problems i have here. I 've been using DNase I in preparation of RNA, the only thing was the DNase I was stocked at -20 for a couple years. could that be the reason? 

Thanks

Yani hu... Hi..!! please let me know what is your control? ?

Hi Shruthi, 

Thank you, I was comparing a knockdown gene cell line and its control. I got the problem solved.

Hello,

I have been having trouble with my negative control showing a band on a gel multiple times while genotyping. My master mix consists of water, buffer, DNTPs, MgCl2, primers, and DNA polymerase. I have run my water, DNTPs, 5x Flexi Buffer, MgCl2, and primers separately on a gel and they show up clean. When I run my master mix without GoTaq polymerase, it shows up clean. The same result occurs when I run my master mix without primers. However, only when I have my polymerase and primers together in the master mix (same as my negative control), a 200 bp band shows up.

I've attached a picture of my setup. Any suggestions?

Thanks for your help.

Albert

also use filter tips

I have the same problem with NCT having CT values and showing a band same size as my positives. I have bought new primers, new water, new syber green kits but i still have the problem.