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A Quick Guide for Troubleshooting Problems with PCR Amplification

Mar 01, 2011
Suzanne Kennedy

PCR is one of the most common techniques performed in virtually all molecular labs today. It is so routine, that when something goes wrong, it can be exceptionally frustrating.  No one wants to spend time troubleshooting a problem that is as simple as mixing a few solutions together in a tube and putting it into a machine.  We need fast answers so we can go on with our research.

Recently in our labs, we encountered unexpected problems while doing qPCR and PCR experiments. As a result, we were reminded of some valuable lessons. I would like to impart them to you here today along with additional advice for troubleshooting PCR problems that usually crop up when you least expect it.

Before I begin with my story, I should explain that we use the Kapa Fast enzymes. We love these enzymes because the PCR and qPCR kits finish in about 45 minutes and the enzymes are so robust that they produce more PCR product compared to other enzymes we’ve used. So of course, getting a negative PCR or low PCR efficiency is never a problem.

End-Point PCR

Let’s begin with mystery of the failing 16S end-point PCR.

With end-point PCR and a 2X Ready-Mix, there’s not much that can go wrong. As long as the primers are added (they were) and they are fresh (they were brand new) and others were using them with success (they were), then I can rule out the primers.

And fortunately, I had some controls in my experiment. I was evaluating a panel of different soils and was working with a difficult agricultural sample type that appears to have a lot of fertilizers or chemicals present, thus always gives weak amplification in PCR but should amplify without dilution. Included in this run was positive control DNA from a soil that always amplifies and it worked as it should. So I was able to rule out the enzyme kit as the problem.

So what’s left? Well, while standing by the thermal cycler, watching it begin the hot start, I noticed it was going into a 10 minute hotstart. I thought it was strange since the kit is “fast” and it needs only a 2 minute hotstart. And then after the reaction was finished, I noticed other changes to the protocol. The extension cycle should be 10 seconds but it was reduced to only 1 second.

The answer to this mystery?

Someone had changed the saved program for our Kapa Fast PCR run and forgot to change it back. I changed the program back and all the samples worked as they should.

You would think that because the PCR worked for some soils but not others, that the program wouldn’t matter. But it does. Apparently, when the sample is difficult to begin with, having the cycling conditions just slightly not optimal can cause negative results. With control DNA it worked fine.  The morale of this first story is: when your PCR stops working, check your machine and make sure someone didn’t modify your program.

I should note that our typical advice is to dilute the samples 1:10 when they do not work undiluted and this always amplifies. But I was using a soil I know works undiluted so I couldn’t rest until I had it right.

qPCR Troubleshooting

Around the same time, we were faced with our first ever qPCR assay that did not work. We tried everything from re-calibrating the instrument, re-ordering primers, to running an assay that works in the machine with the chemistry to show that the enzyme and machine were not to blame. Then we also tried adjusting the annealing temperature, time, and extension time. Nothing worked.

The melt curve data was most informative. We could see that the amplification was not specific. There were multiple curves in all the reactions.  When this happens, it is usually a poor design of the assay.

So we went back to the original paper that we took the assay from and sure enough, when we looked at their data a little more closely, their efficiency data wasn’t very good either.  They didn’t show melt curve data but we suspected the assay worked the same for them as it did for us: poorly. But it was published anyway.

The lesson here is: when you take an assay from a paper, check that they reported all the necessary information according to the MIQE guidelines. Researchers need to give full details about their qPCR assays including the PCR efficiency and sensitivity.

We found another assay for the organism that is giving us the 95% PCR efficiency we are used to and the melt curves show only one peak.

Sometimes it’s not always a bad thing when a PCR fails. If we never had to troubleshoot, we would never learn anything.  Here is more advice for troubleshooting PCR problems.

10 Tips for PCR Troubleshooting

1. When working with an existing assay, always have a positive control (and a negative control) so that you can rule out a problem with the primers, enzyme or a machine setting. Check your program and make sure it’s correct.

2. When designing a brand new assay and testing it for the first time, include a positive control reaction so that if the new assay fails, you know you should focus on the primer design and not the chemistry.

3. If the PCR products appear as a smear, you may need to increase the annealing temperature or decrease the magnesium (if you added Mg yourself and it wasn’t already in the mix.) You may also be adding too much template.

4. If your amplification is weak or non-existent, many things could be happening. Dilution of the template 1:10 will let you know if the issue is a PCR inhibitor.  Try diluting the DNA first if this is an environmental sample. If not, try bringing the annealing temperature down a couple degrees or adding additional magnesium. Conversely, the template may be GC rich and you may need a longer hotstart or an additive to help melt the template.

5. Make sure you are following the protocol for your kit, including the amount of time it needs for enzyme activation and the cycling times. Each manfacturers kit is different and optimized for their chemistry.

6. Check your DNA template on a gel AND a spectrophotometer or with picogreen. Don’t trust the reading alone.  Make sure you have DNA (and not RNA) and that the yield looks accurate to the Nanodrop or picogreen reading.

7. With qPCR, set up 10 fold dilutions of template for the standard curve and use at least 5 dilutions to have the best sensitivity and linearity. Your assay is only accurate down to the lowest Cq that you can detect that is linear in the assay. Once the assay loses linearity, those sample past that point cannot be accurately quantified. A perfect assay will have a slope of -3.3, meaning that every 10 fold dilution is 3.3 cycles higher. This is 100% doubling in each cycle.

8. Some SYBR Green kits use three step cycling (denaturing, annealing, extension) and some use two step cycling (denaturing and annealing/extension combined at 60C). Follow the directions for your kit.

9. When you open a new enzyme kit of a different lot or get new primers, repeat the standard curve again. Make sure you get the same Cq values for the same dilutions.  If the primer synthesis was poor, you’ll be able to catch it right away.  If the curve is different, you will be able to calculate the data correctly and avoid misinterpretation of the results.

10. Signs that the primer design is a problem are mutiple melt curve peaks, non-specific amplification, and poor PCR efficiency. On an agarose gel, the assay should give you one single band.  It may be the oligo synthesis but usually it is the design. There are a number of PCR additives you can try that may help if you have no choice but to design an assay in a troublesome area for polymerase.

NTC contamination issues:

No template control problems affect everyone at some point in their PCR career. I wanted to devote extra attention to addressing this annoying, but common problem. Here are some possible reasons for PCR contamination and solutions for solving the issue.

1. If you are amplifying with 16S primers, the contamination is probably coming from the enzyme. This is very common and difficult to avoid.  If this is a gene specific primer and you have contamination, then it may be true contamination of a reagent.  Here is what we recommend for eliminating the chance of false positives:

2. Designate a separate area of the lab as a PCR station and do not use it for anything else. Ideally, this is in a different room than where the DNA is prepped and PCR products are handled and analyzed.

3. Purchase a set of PCR only pipettors and do not use them for anything else.

4. Wipe down your PCR area and pipettors with a Lab Cleaner that removes nucleic acids and follow that with wiping the surfaces down with 70% ethanol to remove the cleaner.

5. Always use aerosol resistant tips.

6. Keep clean water in the PCR only area for use with PCR only.

7. Add the positive control DNA at your bench, after the NTC reaction has been closed. Do not bring your test samples to the PCR area.

8. Aliquot your primers and your enzyme mix if you purchase large volumes. If something does come up positive, you can always throw away the small aliquot and grab a fresh tube. This way you don’t need to throw out an entire kit or batch of primers.

******

Read on...

143 Responses to “A Quick Guide for Troubleshooting Problems with PCR Amplification”

  1. Shahid says:

    I have trying to perform a PCR . the target belongs to a multigene efamily so basically i am getting multiple bands in my PCR reaction. i tried iptimizing by altering concentration of magnesium chloride but i still didnt get specific bands. also im using cDNA as my template. the cDNA is alright as i have already tested it using a housekeeping gene primers. any suggestions?

  2. Pooja says:

    Hi,

    I am trying to amplify 7 genes from past 2 months from bacterial genome but they are not showing any amplification…please give ur valuable suggestions

    • upasna says:

      1) check ur dna sample,primers (blast the primers aswell) to check its specificity.
      2) do a gradient pcr with undiluted and diluted dna

    • suchit says:

      decrease annealing temperature.and also change primer concentration as well as manage MgCl2 -2 to 4 mM

  3. Tito says:

    Hello, I am currently facing a problem regarding PCR. I am amplifying a 650bp DNA fragment. On the gel I find a predominant band at 400 bp and a little bit of 650bp (as well as other smear like bands). I carefully cut the 650bp band and gel purified and ran the sample again on the gel, interestingly I still find a predominant 400 bp band. I wonder what could be the problem. If anyone can help I will appreciate a lot. Thanks for taking time to read this.

    Tito

    • Ashley says:

      I have the similar problem to Tito. Did you ever work out wat it was? I am amplifying a 434 bp band of bacterial dna so I clean it up cut it out then run it on a gel again and it gives me an additional faint 1000bp band.

    • Jon says:

      When you run a gel, it is not a perfect separation mechanism. Especially with smaller fragments. You still have some of the smaller fragments stuck with the bigger ones. Smaller fragments are preferentially amplified during PCR – especially if your primers are also preferentially binding to the smaller fragment then it is doubly so.

  4. DUA says:

    hi

    i am performing an tetra primer ARMS PCR using 4 primers 2 outer and 2 inner .the problem is that i am not getting the band from 2 outer primers shows gene segment?while getting the band shows polmorphism .Can anyone help me out what was the problem with my PCR??

  5. malvika says:

    hi! i am just curious to know that what happens if the amount of water added in PCR mastermix is more? For instance, i accidentally added 100ul water more to already existing 538ul water for 100 reactions. The primers,buffer and dNTPs are 100ul each though. will i have any issues in my result?

    • Jon says:

      Your buffer concentration will be more diluted then the optimal working concentration. This will decrease the concentration of your salts which can affect the annealing temperature of your primers as well as alter pH in such a way as to render the enzyme less effective. Depending on how much you diluted from 1X it may or may not change things too much. Only one way to find out – run it.

  6. Steve says:

    Hi Everyone, I’d recommend everyone a PDF material I’ve found that you can get from pcrguru {dot} com. There are tables of additives and various troubleshooting options depending on the problem you get. Really neat stuff.

    • Sonali says:

      Hi, I am also facing same problem with pcr amplification I am getting smear on gel, with 6 SSR primers pair. as a template i used more diluted dna but it does not work. I got amplication with same diluted dna with one primer but other primers giving smear, sometimes no bands. From two weeks i am facing this problem, i have to complete my research early. Please help me…..

    • Emelia says:

      Feel free to email technical@mobio.com or give us a call at 800-606-6246 :)
      We are here to help!

    • suchit says:

      increase annealing temperatur and template concentration

  7. Dr Kishore says:

    in spite of repeated trials with primer concentration, mgcl2 and template i am not getting any bands with my ap-pcr for oral anaerobes. please advise how to go about it.

    • Emelia says:

      Hi Dr. Kishore,
      Have you run any positive controls? If your positive control is not amplifying, this means there is a problem with the PCR. If it is easier, feel free to email technical@mobio.com or give us a call at 800-606-6246.
      Sincerely,
      MO BIO team

  8. Nivedita says:

    I am trying to amplify a vector for cloning, it is about 7kb in size. Each time I perform a PCR and run the product on gel, I see nothing but weak smear. Before this I was trying to amplify 11kb plasmid, which did not work out at all. I don’t understand what could be the problem. I have decreased the primer amount and changed cycling conditions as well. can anyone help, please???

  9. akshi says:

    hi
    i am getting amplification in my negative control.i have changed all the reagents one by one (MQ, buffer,dNTP,primer ,enzyme) but still i am getting amplification. can anyone help ???

    • Michelle Tetreault Carlson says:

      I’m sorry to hear that. Since you’ve already changed out the reagents, I’d suggest looking at external causes. For example, it could be that your pipetters are contaminated. It could also be due to aerial contamination during the preparation of the plate. Try cleaning everything with bleach and ethanol. If your amplicon is small, <100 bp, it could also be that you are seeing a primer dimer rather than an actual band.

  10. Cat says:

    Hi. I also have a problem with qPCR. Specifically with amplification of standard dilutions produced from plasmid (environmental samples, from soil, are amplified with no problem). Only 2 dilutions (10^9 and 10^8) are amplified. When the dilution set was tested on regular PCR, only the dilution 10^9 is amplified (both were checked on gel). Which suggests product degradation. However, other users working with different genes (and different protocols and reagents) are also experiencing the same problem. Because in both qPCR and PCR we are unable to amplify it excludes faulty instrument. I wonder if there’s something that originate degradation during dilution preparation? Any ideas are welcome! Thanks!

    • Michelle Tetreault Carlson says:

      Hello Catarina,
      So what you are saying is that you only get amplification when the template is diluted to a high degree. For higher copies of plasmid you aren’t getting amplification. You are only seeing this with plasmid and with nothing else? First, make sure that this is linearized plasmid that you are working with. Did you check it on a gel to make sure that it has your insert? It’s possible that some PCR inhibitors are being carried over from the plasmid prep. You could check this by adding a small amount of your low dilution plasmid to your positive control. See if there is something in the plasmid solution that inhibits the positive control. Feel free to send images of your data to technical@mobio.com
      Best,
      Michelle

  11. Rehab Mohammed says:

    I’m detecting three genes, the expected band size 142, 137, 200. I’m using positive controls first to djust the primers conditions but the positive controls show no band while negative controls give bands with the exact size. I repeat the test and the same result appear

  12. Rehab Mohammed says:

    I’m using three primers for detecting 3 genes. the expected bnd sizes are 140, 137, and 200 bp. while adjusting coditions using positive controls (colony PCR), unexpected result, no band with positive controls and sharp bands with negative controls at the exact size. repeated test give the same result. can you help me to find the problem?

  13. hi says:

    Hi?

    I had mixed my template DNA and primer and kept at 4oC overnight. If I use this mixture of primer and DNA, is there any problem for PCR?

  14. Cat says:

    Thanks Michelle! I only get amplification with high copies of plasmid. Once it becomes more diluted i am not able to amplify it. I did check on a gel both in normal PCR and qPCR and no band was present for higher dilutions but, the lowest dilution (high copy numbers) presented a band in the correct size which suggests it has the correct insert. It’s been happening with different plasmids with different inserts, more or less randomly. I will check if there’s some PCR inhibitors being carried over from the plasmid prep. Thanks for your suggestion!

    • Michelle Tetreault Carlson says:

      Hmmm. If PCR inhibitors from the plasmid prep were the problem I’d expect the problem to get better as the template was diluted and not worse. And so I’m thinking that this is not the cause. What concentration of plasmid are you starting with and how are you measuring it? A Qubit or e-gel is going to be more accurate than a Nanodrop. Feel free to contact me at technical@mobio.com if you’d like to discuss directly.

  15. Eduardo says:

    Hi:

    I have a problem with a protocol of begomoviruses, and I need to amplify a DNA fragment of 2.8 kb. My primers are universal and I have run 3 thermal profiles, but it doesnt work, I have sometimes fragments of 400, 200, 100 bp in my positive control and target sample. I dont know what I can do.

    I hope that you can help me.

    P.S: my primers were checked in ncbi and they doesnt have problem there were perfect.

    • Michelle Tetreault Carlson says:

      Hello Eduardo,

      There could be issues related to the length of the fragment being amplified. Would you mind answering the following questions?

      1.) How long is your current annealing and extension time? Under absolutely ideal conditions, Taq polymerase can process 60 nucleotides per second, thus for 2800 bases, the extension time would need to be at least 47 seconds. More routinely, however, people seeking to amplify large fragments use much longer extension times (2 – 8 minutes) followed by a 5 – 10 minute final extension at 72 C.

      2.) Are you using a dedicated master mix for Long PCR? If not, several companies sell great master mixes for long PCR which account for controlling pH during the long extension times (pH will change in a Tris-based buffer in a temperature dependent manner leading to more depurinating (acidic) conditions, enzyme combinations (Taq + a proofreading enzyme) or unique recombinants (e.g, Phusion polymerase), magnesium and dNTP concentrations. The attached pdf describes the Eppendorf TripleMaster system and compares it to other kits that are out there.

      3.) If you run no template controls with your primers are you seeing any evidence for non-specific product formation by gel (or better yet, Sybr Green qPCR)?

      Best,
      Michelle

  16. Anusha says:

    Hi,
    I’m doing a multiplex PCR with DNA extracted from human saliva. I changed the primer concentration, DNA concentration, annealing temperature, even checked for contamination to optimize the whole thing.

    My band for exon 4 was showing proper results for the normal PCR, but when I incorporated the Internal control for the multiplex PCR, no IC bands showed and the Exon 4 band became very faint….
    Very confusing as my band for Exon 2 showed proper results on both PCR, and even showed the IC.

    Moreover i checked for contamination of all my reagents, nothing showed on the gel except for primer dimers of the primers… But my NTC are showing bands and my Internal control only is showing more than one band!

    Can someone help me out?
    Thanks, Anusha.

  17. Dr Khin Thin Yu says:

    Hi,
    I want to know some problems concern with my PCR condition. I have already got significant band with respective
    PCR product last two months ago. But now, I can’ t get the same
    band at this site with the same sample and same primers. PCR
    product size was 372 bp but it was seen as 150 bp size on gel electrophoresis. What can I do? Help me, please.
    Thanks.

    • Michelle Tetreault Carlson says:

      Hello Dr Khin,

      Sorry about the delay in getting back to you. I think it’s very difficult to say what the cause of PCR failure is without more information. If the PCR worked two months ago and doesn’t work now then my first guess would be that one of the components has degraded by a nuclease. Have you rechecked the integrity of the primers and template on a gel?

      Best,

      Michelle

  18. RAVI PATEL says:

    HELLO, I am facing problem with pcr amplification, i used PCR Super mix Components:
    22 mM Tris-HCl (pH 8.4), 55 mM KCl, 1.65 mM Magnesium Chloride, 220 µM dGTP, 220 µM dATP, 220 µM dTTP, 220 µM dCTP, 22 U/ml recombinant Taq DNA Polymerase, and stabilizers.
    and use rbcl R & rbclF primer.

    made 20ul system:
    10ul supermix
    1 ul F primer
    1ul R primer
    1ul template DNA
    x ul water

    i run 15-20 times PCR. BUT product was not amplified.. what is the reason behind this problem, i dont understand… So please help me, what i do,,,

  19. RAVI PATEL says:

    and also face primer dimer problem.

  20. majid says:

    Hi dear
    I have trying to perform a nested PCR and I have positive control. but I didn’t any band in gel electrophoresis. help me please.

  21. MGSD says:

    Hey, I have a problem with my NTC, is contaminated apparently… i´m sure that does not is the water or dNTPs, may be the primers?! I did a gradient already. :( inclusive a high temperatures of annealing (68°C).

  22. hi says:

    Sir/Madam,
    I have been doing qRT-PCR for the past few month. I am struck with a problem. I get amplification in NTC and RT negative as well in few genes(No cDNA instead i use RNA).

    I used 1ug of RNA as a template and reverse trancribed them to cDNA using SSII from Invitrogen. Then i ran qRT-PCR for two genes. In one gene i got a specified product in the positive(cDNA from MCF-7). No amplification was found in NTC and RT negative as well. The melting curve was good as well. But in the other gene, the scenario is entirely different.I got ct as undetermined in positive control.I didnt get a peak in melt curve as well(its Tm is 72 & 83).I got some peaks in NTC and RT negative. There is a peak in melt curve(Tm is 81 & 83).To confirm this i ran it on a gel. I had band in NTC and RT negative.The bands are below 100bp so i suspect it to be a primer dimer.Both bands are of same size.

    My question is why do i get peaks in NTC and RT negative? Say if my reagents or primers are contaminated i should have got peak in positive as well right ? If the product in NTC and RT negative are primer dimer, what is the ideal Tm for primer dimer in melt curve?

  23. Joseane says:

    Hi,

    I am trying to amplify the mitochondrial gene (ND6) and nuclear (ITS 1) from past 3 months from fruit fly genome but they are not showing amplification to almost all sample…please give your suggestions. Some sample working to gene, but the same sample not working with other gene.

  24. Kate says:

    Hey there

    I have a problem with my PCR, which is really annoying. I performed the same PCR a few times and sometimes it workes and the other times not. I’m performing a seminested PCR with patient samples and the PFU enzyme. The primer where tested on a gradient PCR which gave me a good band on the right sizes, but with patients they don’t work.

    Regards,
    Kate

  25. Noe says:

    Hi!
    I’m now trying to identify stable genes to use as reference for my expression analysis experiments. The problem is that most of the times, when I do a qPCR experiment I always get some missin values, within my technical replicates, but not in all of them. I mean that for a certain sample, I prepare a bulk with the Master Mix, primers and cDNA. I mix it before pipeting on the plate and then I split the bulk into three different wells on the plate, using an electronic pipet. And the problem is that sometimes I got maybe two replicates with a good Cq value and then the third one without an signal…how can this be possible?? I’m starting to think that it can be the machine…is it possible that the “lecture” of the plate is not being homogeneous??
    Thanks

  26. divya a v says:

    divya av
    am getting more than three bands in ssr – pcr amplification in one sample always. why is it so? please give your valuable suggestions

  27. Brittanie Collinsworth says:

    Due to the popularity of this post and the variety of questions we would like to refer you to a few websites that might be able to assist you with a solution to your problem.

    http://www.researchgate.net/

    https://groups.yahoo.com/neo/groups/qpcrlistserver/info

    We still encourage the community to comment and reply as we love to see the community interaction, but thought additional resources would be appreciated for questions requiring a more urgent response.

  28. Nino says:

    hi, I faced strange problem, with different primers run with same DNA I got 100% identical bands. what could be the reason I wonder.

  29. Anusha says:

    Hi,

    I’m doing a multiplex PCR for exon 2 and exon 3 for the HLA-B*5801 gene. So one reaction is the internal control (IC) and primers for exon 2 and another one would be IC and primers for exon 3.

    I started and got both bands for IC and exon 2. But when I did repeats 3 times after that, the IC band disappeared completely!!!

    With exon 3 primers, the IC band doesn’t show up at all! I’ve tried doing a gradient multiplex PCR for both IC and primers for exon 3, and still nothing… I’ve also increased primer concentration for IC and template DNA concentration too. Still no IC bands.
    What do you think is the problem here?

    I thought maybe the problem was with the IC primers, and I ran a PCR with these primers alone. I get the proper sharp and clear band at 400bp.

  30. Namrata kumari says:

    i am unable to remove dimers in multiplex pcr

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