Hi,

It seems that your upper band is a not-specific one.

I would increase annealing temperature to avoid it. Which is your primers melting temperature? Normally, I design primers at 66 degrees and I amplify at 60 degrees. I would suggest you to increase annealing temperature of at least 3 degrees.

Hi,

It seems that your upper band is a not-specific one.

I would increase annealing temperature to avoid it. Which is your primers melting temperature? Normally, I design primers at 66 degrees and I amplify at 60 degrees. I would suggest you to increase annealing temperature of at least 3 degrees.

Hi ..absolutely....even i consulted wid ma lecturer.. He also said abt the temp factor..

Good to see you got those primers up and going :)

Do a gradient PCR and see if you can't make it more specific. As the non-specific band is above your specific one, shortening the extension time can help too.

Cheers,

Steve

There is also a possibility, if tweaks to the PCR conditions don't solve the problem, that it is still specific amplification, but you may have sequence length variation in your target amplicon. It might be worth the time to look for this by "BLASTing" your gene against the appropriate database/s and examining the multiple sequence alignment for gaps.

Hi everybody,

I agree with you all. Alexandra, if the downstream application of your PCR is affected by the non-expected product, you will have to do a systematic effort to get rid of it. First, follow Kevin advice and blast your primers, is it DNA-PCR or RT-PCR? If it is not an specific product, then you can titrate magnesium (you do not give us information of your PCR conditions, despite it seems that it is quite specific, except for your sister band). You can rise the annealing temperature, short your extension time and/or add DMSO (for instance) at 1-5% (can try up to 10%). But you will have to it step-by-step to avoid losing yield. In my experience, get rid of this sharp longer products is quite tricky. Wish you luck!!

Hi,

I would agree with Francesco. Try increasing the annealing temperature. I also observe from your figure that the PCR product yield is also less. Try increasing both the annealing the temperature and the number of PCR cycles. That should fix the problem. Just fixed a similar issue recently.

What about an alternative splicing???... your gene has differential splicing???,,,

But I would check the basic,,,

1.- Blast your primers

2.- Increase the annealing temperature

3.- Sequence the PCR product,,, just to be sure!!! : )

Good luck!!! : )

Ya I agreee with most the comments made previously..

Try to Increase annealing temp to lose the extra band..

if that doesn work try varying the MgCl2 conc, as even very small variations in MgCl2 conc can make a big diff in a PCR's outcome especially in case of reaction volumes as less as 25microL..

You may also have ssDNA migrating slower than dsDNA. It maybe caused by the assymetric nature of your PCR (one of your primer annealing better than the other)

Increase your Mg concentration. Mg is critical for selective amplification.

Pls check your primers can amplify only region or two.. I think your PCR condition is ok but the primers specificity must check.

Cheer!!

HI ,

It can be non specific band, do the PCR with higher annealing temperature , decrease THE extension time and template concentration. You can take advantage of gradient PCR and optimizing magnesium concentration in the same time . it’s better u trace different optimization and your experiment step by step.

==>Try additives:

1)Formamide :increases the stringency of primer annealing, resulting in less non-specific priming and increased amplification efficiency. Concentration range: 1-10%.

2)Tetramethyl ammonium chloride: Similar action to formamide. Try out concentrations from 10-100mM.

I agree whit francesco, alternatively you can decrease MgCl2 concentration.

I think you may do a restriction digestion first to minimize your template size so that the primers anneals more specifically ..

Hello Dear All,

I agree that bands are non-specific, but as i think sometime primer dimers takes place due to non specific - contamination... this may be of gel-tank, taq- buffer, PCR buffer or something else, please do one more time your whole experiment with new PCR buffer i think your result will change............and one more thing please change your 1% TAE buffer in your gel-tank and use biomarker to know about the specificity.........keep smiling and do your best. Good luck !!

Hi,

I agree with all of you.

Apart from this, It is not clear if you are using DNA or cDNA as a template for PCR!

1. If it is cDNA and the target gene you are trying to amplify is having introns....Your cDNA is carrying DNA contimation. Go for DNase digestion of RNA and then proceed ahead for RT.

2. Sometimes due to alternate splicing you may get more than one gene product, which get amplified.

3. BTW what kind of primers are you using? Are they specific or degenerate. Usually we get non specific amplifications along with desired when using degenerate primers. Increase annealing temperature or try other primer pair.

2.Try other options like increasing annealing temperature, decreasing extension time,Set up a gradient PCR.and so on. There are many suggestions already.

Also primer dimer is not a problem here. You are getting amplification.

If you have PCR product...dilute it to 50, 100 and even 250 time. Then set up gradient PCR considering other factors suggested by others.

All the very best!

Hello,

I agree with Marcell Nikolausz : this looks like single strand DNA. The amplification may be specific but one of the primer is more effective than the other. A change in the annealing temperature either up or down could eliminate the artefact. May be you could also try to increase the annealing time. may be try also to change the relative concentration of the primer. It may also be that one of the primer is not at the right concentration or not pure enough It must be noticed that if this is indeed single strand, there is a considerable amount of it, since ethidium bromide is a lot less effective at binding single strand DNA than double strand DNA.

I agree with Nokolausz and Bonnet. The ssDNA product.

Dr Mitton,

1) the part of cyt b of fish mtDNA

2) The company that made the primers pointed Tm 50. I amplified at 48 (the nedative result) and at 50 (non-specific bands, as you can see). But the authors in article use the same primers and they amplify at 55 degrees.

3) total DNA

4) lane 1 is one of the sample I amlifiead for the first time as well as others

Hi， your primer may not be specific to your interest band， first you can increase the annealing temperature or conduct a temperature gradient PCR. if the nonspecific band still exsit, you should think about designing primer again.

primer is not specific but contamination in your 2 4 6 7 9 PCR tube.

You may cut the band out and make PCR wiht this DNA using your primers. If you get two bands again, the reason is probably ssDNA, if you get one band then your primers are unspecific.

Hi I think your design primer attached two region in your sequence

P.S. no, you have then two bands anyway...... so better seqense your unspecific band)

Dear Alexandra

Please send to me your primer sequence, PCR program and target sequence for better analysis.

Try nested PCR

mt DNA doesn't include introns，so you should adjust the annealing temperature ，or redesign the primers。

Hi,

The uper band is not specific. You can add MgCl2 to your PCR or increase annealing temperature, and add five to ten minutes of final elongation at the end of your PCR programm.

Good luck

Thank you for your comments!!! You all are super! I achieved a positive result following your advices!!! What should I do, if you didn't help me? ;)

What did you do ? What was the problem, finally ?

I would also like to know the final result. To follow the branstorming is really edifying, but I always miss the conclusions. Dear Alexandra may I ask for some brief final report?

I agree with Francesco Fiorentino

You need optimization of PCR. You can number of thing to remove the nonrequired band.

1. Do a gradient PCR and see if you can't make it more specific.

2. manipulate with primer concentration once you calculate your near ideal Tm for your template in this experiment.

3. You can change temperature of annealing time along with combination of ramping effect to remove undesirable non-specific band.

I hope these experiments will provide you good specific products

Ahsan Sheikh

If you used primers for amplification of ITS is normal appear multiple bands on gel

Diogo Pinho

Change the anneling temperature for high, probably these double bands will desapier!!

good lucky

it looks like unspecific amplification, you might be getting 2 PCR products instead of one. When you designed the primers which was the expected product size? Did you blast the primers before using them?

Well, I used an annealing temperature calculator, according to the sequence of my primers. the calculated Tm was 6 degrees above the temperature specified in the documents of the manufacturer. So, according your tips I changed the anneling temperature for high and these double bands desapiered! What is the conclusion??? Never believe a manufacture, always check :)

The two bands in 7th and 9th line seem alike

You could try varying your primer concentrations and also do a gradient PCR with different primer concentrations. It seems like there is excess primer in your reaction mix which is why you are seeing a bright bands which maybe excess primers and lighter bands that are non-specific bands. What is the molecular weight of these bands ? are they down the molecular weight ladder or higher up ?

It is always better you try both methods,calculate the annealing temperature on your own and also try the manufacturers instructions. Standardize by varying parameters according to the results you observe

Why not try adding 10 % BSA in you master-mix.

If all your lanes have the same product as it seems to be then why 1 is different? If the conditions/parameters for each tube were different then you can run another gel with smaller/lesser amount of DNA to see the band clearly on the other hand If the parameters were the same i would prefer to do a gradient PCR before playing around with some other variants. Its always good to do a gradient PCR straight away with new primers as it optimizes your PCR in one go and saves a lot of time at the end of the day.

hello, I´d got the same problem and I increased the annealing temperature but my really solution was decrease Mg concentration.

My adviser, think so Raibel Suarez. About Tm.

And depend on yours aims, you would like to known?

Hello

I think your primer is not so specific and reproduce two region you can chang your primer or annealing temperature

You can do a hotstart at the same temperature or try out reducing the Mg conc

non specific band is coming check once annealing temp and check the GC content with in desired product

Sorry...but where is ladder lane??? Is important to understand if your bands are really a double bands, like primer dimer.

Hi. Check the mt of each primer and the annealing temperature. The annealing temperature must be lower than the mt. What is the PCR program?

Ladder is important to define PD bands, where is your? What is the fragment size? Besides takes time T gradient PCR is the best choice.

Hope your PCR problem is solved. Other wise as Nelson mentioned DNA ladder is always needed. First you should know your fragment size. Then play with annealing temp. Certainly it works. Primer dimer always come below 100 bp. In your case it is not primer dimer. When you design the primer it gives Tm, melting temperature. In general Ta, annealing temperature always should set 5 degrees less to Tm.

it would be easier if you can give more information on what you are trying to amplify. As most of the comments above I suggest you to run a Temperature Gradient PCR and also to do a BLAST search to verify the specificity of your primers. Using the ladder to confirm the correct size of the band is also necessary.

did you check the denatured dna band hypothesis ?

If it iscorrect, by just denaturing your PCR product before the gel, you should get one, two bands, or three bands ; one of them migrating like your mistery band, the second, corresponding to the other strand and the third at the renatured product

Change anealing temperature by doing gradient PCR

Dear Alexandra,

I agree with the comments of Santhoshi Rani and Joao Gatica. However, these double bands can also be result of alternative splicing (if this picture corresponds to a RT-PCR), i.e., the same primer set can amplify similar but different size mRNA. I don't know which organism you have been working with but alternative splicing is very frequent in insects. In this case, alternative splicing would be an interesting found.

Anyway, to check it out you will need to sequence both fragments.

Best wishes,

Adriana

I agree with Nelson , where is the ladder, and what is the size of the fragments you detection of them, and the upper bands that no specific, and Good luck

Dear Francesco Fiorentino.. i am also facing the same problem trying to clone 3 target genes of my interest. I used the genomic dna as template to amplify using Phusion DNA polymerase. at the first got the amplification but as i got the amplification in the negative control (water) which i never saw during my first PCR using Phusion. then i tried again using dd water in a fresh bottle and cleaned pipettes.. this time i cud not get negative control bands but i cud get high and low olecular weight bands of my target amplification. i am trying to clone 2.2, 1.9 and 1.8kb of insert in the T vector but i want the amplification.... i ll atatch the pics ... please suggest me. i tries different range of temperatures... lane 1 is genomic dna with size 1.9 kb, 2 is water negative control, 3 is the plasmid positive control. "S48 -58" are the range of temp for amplifyinf a fragment od 2.2 kb and i cud consider the lanes for 50 to 58 temp as right.... but still i am confused wheni tried doing for higher volumes (40 microliters). but for the higher vol i used 2 microliters as the template (the same as for the 20 microlit). pcr and pcr 1 were my first attempt. next day i repeated with high volumes 40 microl. i am sorry i think i cant attatch multiple images????

Francesco Fiorentino again.....

my todaya attempt... usong higher volumes but buffers dntp and primer same as i used in the before experiments.

i thonk may be i used less genomic dna .. but still it may not be a problem ... i tried to increase the annealing temp and cycles.. but why...cud u suggest me.. Francesco Fiorentino..

well.. others dont mind to give me suggestions .. reason being specific is jus i saw his reply first..

u r genius Francesco Fiorentino...

Cheers

try to read this one in the link

http://www.bio-rad.com/en-us/applications-technologies/pcr-troubleshooting?ID=LUSO3HC4S#gel2