PCR smearing trouble shooting
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PCR smearing trouble shooting
We have been getting a lot of smearing in our PCR reactions. Sometimes we
get a discreet band, but most of the time its a smear. We get this with
different primer pairs so it dosent't appear to be confined to one pair.
The reactions are all set up on ice to minimize aberrant priming before
thermalcycling. I would be grateful if anyone could give me information on
how to eliminate the problem.
Phil
Newsgroup archive
Is this RT-PCR?
If so you could try an anchored oligo dTX or dTXX in your RT step.
Cheers
Bob; Sunny Scotland
Newsgroup archive
What kind of PCR reaction is this, e.g. what are you trying to amplify,
what is your template material? Have you tried to vary different
reaction conditions (annealing time and temp, primer concentration etc)?
Have you tried touchdown PCR?
Cheers,
Malte
Newsgroup archive
Too much enzyme - should be 1.25u per 50ulk but note a lot of unlicensed
enzyme may well be 5x that i.e they calim it is 5u/ul but it could
really be 20u/ul etc.
Too many cycles
Contaminated pipette barrels/water/buffer/dNTPs etc.
Go back to square one. Fresh barrier tips and bleached pipette barrels.
Take M13 universal and reverse sequencing primers and PCR 1ng for 15
cycles max of some simple small insert, maybe even just the polylinker,
in a pUC based plasmid. Use fresh reagents and if required run a
dilution series on the enzyme i.e. set up an initial 100ul PCR, add 2.5u
of enzyme, mix, remove 50ul to another 50ul PCR without enzyme, mix
repeat 4 or 5 times. Use 50C annealing. Does it work without smearing?
If OK move to genomic and try again.
Duncan
Newsgroup archive
My first reaction to your e-mail was that your annealing temperature during
PCR is too low. So you may want to try a little experiment testing a range
of annealing temps (a range of 10 - 15 degrees, increasing by steps of 3
degrees). If annealing temps are too high, the fragments will start
disappearing, if the temp is too low, you get a smear.
I am not sure what polymerase enzyme you are using but after setting-up
your reactions on ice maybe you could try a preliminary step of 2 min @
94*C at the beginning of your PCR. This should not harm your enzyme but it
would ensure total denaturation. Also PE Biosystems makes a polymerase
called Amplitaq gold that can be added to the master mix at RT and then
requires a prelim heating of 10 min @ 94*C before the cycling. It is
probably more expensive than otehr polymerases, but it works consistently
everytime :-)
Also, did you run a gel (0.8 - 1.0% agarose) with just the DNA to check the
quality of the DNA you are starting out with? You should get a nice tight
band high up on the gel. If you get a smear here, your DNA is of poor
quality.
Another suggestion is to try optimizing DNA & MgCl2 concentrations. Are
you running your fragments out on acrylamide or agarose? Is agarose at the
correct % for the fragment sizes you are looking at? In general, 1.5% -
2.0% should be used for fragments 300bp - 2000bp in size.
Hope this helps
Deanne Bell
Newsgroup archive
Last update 22-Mar-2002, Rating Fair of 18 votes.
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For getting your product back, try lowering the annealing temperature a degree or two and see if the fragment returns. You might also want to try/re-try a MgCl gradient (trying several Mg concentrations in the same PCR) to see if these DNA samples have altered chemical/protein contamination than the ones that previously worked.
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Reaction was working before, but now I can't get any product
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I do the SOE PCR,, but the smear is serious,i change the Mg ion and annealing temp, but the smear is still,and still no band at all.i hope who can help me! i put the mulitple temple A and mulitple temple B in the pipets thank you
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I do the SOE PCR,, but the smear is serious,i change the Mg ion and annealing temp, but the smear is still,and still no band at all.i hope who can help me! i put the mulitple temple A and mulitple temple B in the pipets thank you
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you decrease the annealing tempereature
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u first conform that all reagents are working well , ihope u r dNTps got contamination ,and till now u r chenging all expect u r template ,chenge u r template and try again,and try with anothe pipets
best of luck
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Imet the same problem with my multiplex pcr on 8-cells embryos but when Ido multiplex on DNA extract of blood or with my a pair of my primeres I dont see smear.so please HELP me.
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thanx for your attention .your response maens that i decrease the Mgcl2 concentration?note that Im working on 8-cells embryos!but I didnt have this problem with 1microL DNA.
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Hi All,
I have the same problem with my PCR. I used the template 50ng/ul. I've tried with vary annealing temperature but it seems no different.. can anyone help me?thanks
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i am getting a nice band but with very fine nonspecific band appears immediately below that band. so how can i avoid this non specific band because that is like attached to that band
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I get a very thick band just less than 100 bp and no band at all at the 1.6 kb (16S rRNA) region which is my region of interest. i loaded 5 microlitre of a 25 microlitre PCR product to check this. please give suggestions
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I've got into the same trouble with you.Though the template is plasmid DNA and worked quite well during the first pcr, the following pcr results are quite frustrating. I still can't see a clear band after
changing the temperature and enzyme.
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This paper provides solutions for PCR smearing problem
Anal Biochem. 2006 Jun 15;353(2):296-8. Epub 2006 Apr 7
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=16626617&dopt=Citation
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I too face the same problem of smear. I did nested PCR. It was possible for me to amplify my product several days back. After a while I am unable to do this. I always end up in a smear including my positive control
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Hi all!!
I designed a pair of primers, and they were working daily greatly during 5 months, giving a bright band. From one day to the following, i got a smear in all my samples with that primers. Iīve changed reactives, new primers, different waters, and even termocicler and i went to other lab, but i got the same smear. Negative sample (water) gives me the same result, the same smear. Could it be primer-dimers? and does someone know why suddenly, with new primers, they produce dimers if it didnīt happen the last 5 months??
could it be because of a change in the syntesis of the primers? Iīve asked for the same primers to other company, could it affect to the stablished conditions for that PCR??
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Hi. I also have the same problem with my RT-PCR. My PCR products are smears on the gel. My first strand synthesis products are also smears. How can I improve my results? What parameters can I manipulate? Tnx.
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there are two reason for getting smear:
1. poor handling (during extraction)
2. high quantity of DNA
you can dilute the amount of DNA at various dilution factor abd you can use the followed dilution factor for other results.
you can also check the extracted DNA by making 0.7% agarose to check the smear.
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i also met the same problem. i currently doing side directed mutagenesis using 2 different templates in one reaction. my mission is to join these 2 template. after about a month trying, i just got smearing.
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high ratio DNA (260/280) more than 3.
what is the usual cause?
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i met the same problem, but i used the bacteria culture cell as PCR templates and M13 as primers. Although PCR optimizing has been done, the problem still there.So, is the culture cell storage period affect the PCR afficacy,or some compounds produced from the cells prohibit from the targets amplified?i will appreciate you if you can give me any helpful suggestion.
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Smear problems in PCR is most usually a bad ratio of Mg concentration vs. DNA (template + oligos), because Mg ions are also bound to DNA. Thus, too much Mg will lead to unwanted non-specific annealing and amplification of lots of things. It is essential to optimize the Mg concentration when amplifying a product for the first time.
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