I'm trying to quantify antibiotic resistances in liquid manure. To get a standard, I cloned the resistances into a pGEM-T vector and extracted the plasmids of viable clones (QIAGEN Midi Prep) . After that I checked the DNA quality using a NanoDrop, and agarose gel electrophoresis. Then I prepared a serial dilution of the isolated plasmids to use them as a standard in qPCR.
For the qPCR, I use the Thermo Scientific Maxima SYBR Green qPCR Master Mix (2x), VWR white semi-skirted 96 well plates, VWR ultra-clear sealing film for qPCR, and the Eppendorf Realplex² Mastercycler. The primers I use have been extensively tested in standard PCR with great results. They have been used in qPCR successfully, but not by me or in my lab.
The problem is that right from the start my plasmid dilutions showed the expected results, but the NTCs were positive, as well. The curves come up around cycle 35 (although that varies), and they show the same peak in the melting curve. The bands in an agarose gel confirm that the amplified fragment is of the same size, as well. So I thought something must be contaminated. I tried it with new primers, new SYBR master mix, new water, new plates, different pipets, with stuffed tips and without, even in another lab with a different clean bench, I had a colleague watch my working process to make sure I don't make any mistakes, I tried it without clean bench, I pipetted the NTCs before I even opened the tubes containing DNA and sealed them until I was done pipetting the samples, and I switched up the positions of the samples in the wells.
In two cases, there was no amplification in the NTC, but it was the usual set up. I used the same pipets, mastermix, primers, water, plates, and primers, I used before (and after) with positive NTCs! I really don't know what do do anymore.
I attached pictures of the raw data and melting curves of two qPCRs I ran, trying to amplify a tetM and tetO antibiotic resistances. The first attempt didn't show a signal in the NTC triplicate, the second did. They were done under same conditions.
Has anyone perhaps an idea what could be causing this?
It can happen that you have a tiny contamination. Some molecules floating around in aerosols. It can happen that one, two or even some more molecules may so stick to a pipet tip being moved through the air or directly flying into the reaction tube. In this case wou sometimes find "positive NTCs", but with very high Ct values (>32). The PCR is sensitive enought to detect (successfully amplify) even a single stupid molecule, when it happens to get into the reaction.
It is next to impossible to get rid of such DNA-contaminations. When my assumption is correct, then you can still work quantitatively as long as you do not want to demonstrate the abolute absence of the target sequence and the lower limit of quantification is some cycles earlier than the lowest Ct value observed for the NTCs. Intead of claiming "the sequence is absent" you can state that the sequence is "below the limit of quantification", what can well be good enough for most purposes.
Good luck!
It can happen that you have a tiny contamination. Some molecules floating around in aerosols. It can happen that one, two or even some more molecules may so stick to a pipet tip being moved through the air or directly flying into the reaction tube. In this case wou sometimes find "positive NTCs", but with very high Ct values (>32). The PCR is sensitive enought to detect (successfully amplify) even a single stupid molecule, when it happens to get into the reaction.
It is next to impossible to get rid of such DNA-contaminations. When my assumption is correct, then you can still work quantitatively as long as you do not want to demonstrate the abolute absence of the target sequence and the lower limit of quantification is some cycles earlier than the lowest Ct value observed for the NTCs. Intead of claiming "the sequence is absent" you can state that the sequence is "below the limit of quantification", what can well be good enough for most purposes.
Good luck!
open yor gilson set and put the white cone and other reagents under UV for 10 minutes to break any pDNA moleculs that may contaminates your stuff. also primers (they are too short to be hitted by uv ray)
I recommend you set up the well plate in a DNA free area, and then add in other area the samples, sterilize everything with UV, including your well plates and the sealing film, and above all ... Do it fast! Best regards Michèle!
You mentioned that you ran an agarose gel and had bands of the expected size for contamination? What is the size of the band that you see? Your melting curves indicate that the negative controls have a different melting temperature (I think, it is hard to tell when you cannot hover over the curves). You have 40 cycles here, how many cycles did you run these primers for during your standard PCRs? Remember that the sensitivity of qPCR may be higher than standard gel methods and by running 40 cycles here you may also see something in a gel that you would not see in your standard PCR. Do not completely set aside primer design issues just because these primers work well under very defined conditions (probably in a different buffer and thermocycler) for standard PCR.
HTH - Becca
I also would suggest using the Taqman mix with UNG (Uracyl N Glycosylase) in order to prevent cross contamination from previous cycles. Are all your NCT amplifying? or just 1-2 out of the three replicates? If all three replicates are amplifying, for both your endogeneous control and your test assay, then I would suspect contamination of your master mix,water, or pipettes. If all three NCTs from just your endogenous OR your test gene amplify, then I would suspect contamination of your assay itself. If the contamination is sporadic, only some of your NCTs, I would suspect your technique and aerosols/pipettes. It should go without saying, but also, use filter tips. Also, follow the recommendation above, or taking the barrel apart of your pipettes and exposing to UV for 30 min. You can also try to set up in PCR hood, if available. Finally, if using the STEP one plus instrument, has the instrument been calibrated lately?
I think that the contamination spread when you cloned your target DNA into a pGEM plasmid.
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