University of Gour Banga
Question
Asked 13 May 2013
PCR product shows a smear in the gel
I loaded a PCR reaction on an agarose gel and I get a long smear with my band a little more intense in the smear. If I dilute the DNA I obtain my band but not perfectly clean from a sample. For some samples the simple dilution did not work. Does anyone have advice?
Most recent answer
I have faced the same issue when conducting PCR with manual master-mix. I have used different combinations for master-mix preparation. Finally achieved the desired result. I have prepared 4 different master-mix set and achieved very good result in set3. I have attached herewith the master-mix preparation calculation along with PCR gel image. Hope this is helpful. I have used Taq polymerase of 1U/ul if anyone uses 5U/ul then use 0.25ul of taq and add nuclease free water accordingly. Thanks.
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Popular answers (1)
Imperial College London
I'm having the same issue also, I am getting smears including my blanks which ruled out the DNA being an issue. However when I did other reactions with different primer sets, these worked absolutely fine. What is also frustrating is this reaction has been done before and worked well! This led me to believe I had contamination in my primer sets so I ordered completely new stocks. I have tried it again and I'm having the same issue. Now I'm moving on to reducing the primer conc but why all of sudden this is an issue when it was working fine before? I'm at a slight loss!
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All Answers (41)
Friedrich Schiller University Jena
Degradation of the target DNA can indeed be a major problem. Letizia and Bartolemeo are absolutely right in this respect. The more short degradation products you have in your DNA preparation, the higher self-priming effects of the DNA itself will be - completely independent from the primers that you add. Normally, however, you see the bands that you want to reveal in front of the smear. In this respect your gel is somewhat surprising for me. Do you primers fit perfectly? Or are you working with complex primer mixes (often called degenerate primers?) - Please follow Bartolomeos advice with respect to controls and in addition check for self-priming.
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Dear
at first make sure about your primer is being not contaminated. More over you can try to make new working solutions with some new reagents. Please Quantify your DNA before use.
Children's Cancer Research Institute
Even if your DNA is slightly degraded in most cases the PCR will still work.Your DNA concentration in the PCR reaction may be too high. Quantify your DNA, and make a serial dilution to determine the optimal amount of DNA for your PCR.
If this does not work try to add e.g. DMSO or use another polymerase or design a new primer pair using Primer blast.
Here you can also find suggestions to a similar question:
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Narsee Monjee Institute of Management Studies
Have you tried quantifyin your DNA example ?? using template dna in low concentration may solve your problem
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Banaras Hindu University
reason behind the smearing is high amount of DNA. Dilute the samples and prepare each and every reagent freshly then try to do again PCR. if it repeated again, then it may be because of degradation of primers.
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All above answers are reasonable, but many aspect can results in a smear. Quantity (template/primers) of course, template and primer degradation for sure. But if any of the abovementioned are right and properly checked, you have to deal with PCR conditions (annealingT, Mg2+ concentration, adding BSA or DMSO to the mix).
I recommend to try a touchdown PCR before starting to deal with mastermix components.
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Shenzhen Institute of Advanced Technology, Chinese Academy of Science
I'm facing the same problem now, I really would like to know how peole solved this, below is my gel image.
Imperial College London
I'm having the same issue also, I am getting smears including my blanks which ruled out the DNA being an issue. However when I did other reactions with different primer sets, these worked absolutely fine. What is also frustrating is this reaction has been done before and worked well! This led me to believe I had contamination in my primer sets so I ordered completely new stocks. I have tried it again and I'm having the same issue. Now I'm moving on to reducing the primer conc but why all of sudden this is an issue when it was working fine before? I'm at a slight loss!
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The University of Edinburgh UK
Dear Yasmin. I am also facing the exact problem, in which the -ve control also showed smearing, Could u please update me know how you rectified it?
Paul Hebert Centre for DNA Barcoding & Biodiversity Studies Dr. Babasaheb Ambedkar Marathwada University Aurangabad
Prepare a new dilution of the primers and new reaction mix with positive one samples then give same result then change the primer set (in this condition your one primer is work but another is not work well )
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Hello Yasmin,
I'm interested if you have solved your problem? Of lately, we are experiencing absolutely the same problem working with degenerate primer pair. We got them two months ago, so it is highly unlikely that they degraded. The final concentration of our primers in PCR is 0.6 micM. However, I found out that Qiagen suggests working with lower concentrations, such as 0.2, and working your way up until you get satisfactory results.
Hoping for the best.
University of Sarajevo-Institute for Genetic Engineering and Biotechnology
Hello Yasmin,
I'm interested if you have solved your problem? Of lately, we are experiencing absolutely the same problem working with degenerate primer pair. We got them two months ago, so it is highly unlikely that they degraded. The final concentration of our primers in PCR is 0.6 micM. However, I found out that Qiagen suggests working with lower concentrations, such as 0.2, and working your way up until you get satisfactory results.
Hoping for the best.
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I know this question has been asked quite some time ago, but recently I had the same issue. What frustrated me was the primer set was giving me a nice sharp bands before, but now my gel only showed smears, without even any presence of a faint--but defined--band.
West Africa Centre for Crop Improvement
Experiencing the same problem as Yasmin's, please update us on how you solved this issue, because it is definitely not my DNA since it works perfectly with other primers,I need help asap.
Universidad de las Fuerzas Armadas-ESPE
PCR smears usually have many causes, most of which have been well described by other authors. But starting with the simplest (following the Ockham Axiom), the problem is contamination. It’s even simpler solution, however expensive, throw everything away and repeat the PCR with fresh reagents. For these cases, is a good habit of the labs, to have aliquoted solutions of throw everything away easily. When the smear seems to appear for experimental reasons, it can be solved by increasing the astringency or specificity of the PCR. For this, add BSA, DMSO, increase the alignment temperature or the concentration of ClMg. If none of this works, it can be DNA degradation or primers
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Huazhong University of Science and Technology
i am having the same problem but it seems no body have good answer. i tried many combination to check primers, DNA, Chemicals. First of all i checked the Primers, i designed two sets of primer for the same fragment. it work well with same DNA. Than i wanted to amplify the whole fragment(worked only for one time) with one set of primer from the DNA as well from the fragment which i amplified with two set of primer ..both shows smear in the gel though i used the same chemical.so i think it is a problem of PCR programs not with the Chemicals, primers or DNA.. Moreover i found that as i increase the size of the fragment, the smear increases...
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University of California, Berkeley
I have been working on solving this for a year and the best solution I've found is to remake the gel if the DNA is new, or re-extract the DNA if it is old. What is frustrating is that DNA fragments that previously amplified perfectly now appear as smears. The best explanation I can come up with is degradation if the DNA sample is old (likely as freezing and thawing cycles) or there are small impurities in the gel/buffer such as microscopic debris that interfere with a clean PCR product. Many times, I have submitted "smeared" PCR products to the Sanger sequencing facility and received a surprisingly perfect sequence. I've also had better results on occasion when using a higher percentage agarose gel (i.e. switching from 1.5% to 3%)
Smithsonian Institution
I see this occasionally and in most cases quantifying the DNA amount (nanodrop, qubit) and using approximately 10 ng total DNA for the reaction fixes the issue. This would be the first place I would start. The smear is all of the genomic DNA in your sample and having too much interferes with the reaction.
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Sichuan University
Hello every one. i need help. I loaded a PCR reaction on an agarose gel . My blank (no template) also show band on specific target gene size. Please help me on this issue. plz find out solution for my problem.
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Swedish University of Agricultural Sciences
its seems that your regents or tools are contaminated. Refresh every thing, hopefully it will work.
Universiti Putra Malaysia
@hanifullah There is a contamination then. I would suggest you to repeat the PCR reactions and use freshly prepared reagents.
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Università Cattolica del Sacro Cuore
Hi Morad,
thank you, indeed I solved by using a lower concentration of DNA and primers and by tuning PCR reaction.
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Tufts University
Giulia Riccioni Could you please explain "tuning PCR reaction". Thanks a lot!
Università Cattolica del Sacro Cuore
Hi Ashwini Sunkavalli, when I say "tuning" I mean carefully select primers, polymerase, buffer, BSA... amount and check reaction temperatures.
You're welcome!
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Tufts University
Thank you again! Did you have to change all the above parameters or was it one or two that helped?
Kafrelsheikh University
so the the possible causes are:
1- templete: high concentration or degredation
2- primer: contamination, high concentration
3- the annealing temperature is not suffiecient
I have the same issue and i will try to assure these points and give feed back
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Hello Dr. Giulia
i suggest you read these:-
1- PCR Troubleshooting
2- Why do I get smeared PCR products?
also It is obviosuly target gene and primer specific but
- Your primers are about 5 x too conentrated: Try 0.1uM particualrly for a small product. This high concentration of primer is evidently encouraging primer dimer formation and that will lead to limited products
- your dNTP and Mg concentrations are fine; as is your DMSO but like the last speaker how did you select your annealing temp of 52C ?
- In truth you can really go down to about 50C in terms of PCR kinetics so I'm hoping that your primers have a Tm therefore of > 55C (Tm - 2to5C is the ideal annealing temp) but I ordinarily would design primers with Tm of 60C to 65C such that the annealing would typically be 58 to 60C
- Have you tried an annealing gradient of Tm-5C to the Tm of your primers in 1C intervals ?, i.e. Tm-5C, Tm-4C tM-3 c etc ?
- In addition with good primers and the correct annealing temp amplification will typically work with 20ng to 50ng of template with a CI of 10ng to 100ng
- In fcat, at concentrations like 250ng ytou can reduce the efficincy of your PCR by overloading with any residual salt in your PCR reaction
- This can be seen as smears on your agarose gel in sample lanes
- Have you tried testing your template with primers to things like gapdh or b actin with standardised condistions to make sure your templates are OK ?
- You could also try testing other DNA extracts that are proven to work with your primers and validated primers to things like gapdh
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Cawthron Institute
Hello, the question was probably already answered, but just in case someone is having similar problems, possible causes are:
- amount of template DNA is too high
- too many PCR cycles (see " Excessive cycling converts PCR products to randomlength higher molecular weight fragments" by Bell and DeMarini 1991)
- low annealing temperature, etc.
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Hamad bin Khalifa University
I have faced the same problem & i just changed the buffer from the running gel and the result was ok.
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Anadolu University
I am also facing the same problem. Can u please let me know how you rectified it?
University of Ghana
1. The primer concentation seems to be too high - primer dimers
2. Play around with your annealing temperature
3. Check the integrity of your template.
Ideally, in a 50ul PCR reaction you want between 20 and 30 ng of each primer - I am also finding that Touch Down PCR protocol is solving a lot of my problems
Pennsylvania State University
I would reduce the extension time and the Mg concentration to decrease incomplete amplification that produces these smears. You should check out this site: https://www.yourbiohelper.com/
University of Gour Banga
I have faced the same issue when conducting PCR with manual master-mix. I have used different combinations for master-mix preparation. Finally achieved the desired result. I have prepared 4 different master-mix set and achieved very good result in set3. I have attached herewith the master-mix preparation calculation along with PCR gel image. Hope this is helpful. I have used Taq polymerase of 1U/ul if anyone uses 5U/ul then use 0.25ul of taq and add nuclease free water accordingly. Thanks.
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