@Dharmesh Jain

Do not change all the parameters at the same time. Change one parameter at a time and check your band. To get rid of the non specific bands, increase your annealing temperature. What is your current annealing temperature?

Is this isolate is different? Maybe the DNA is degraded, try to check integrity of DNA on the gel.

Having a positive control will provide you with more information whether those additional bands are non specific and therefore seen across all samples.

Like other people said you are amplyfing non-specifc products or your primers are hybridizing with each other (primer dimers, which will appear as a low molecular weight band). If by some reason your target sequence is present in low quantity in that particular isolate, the primers will be more available to bind unspecific regions. The first thing I would try is increasing the annealing temperature by a couple of degrees during your PCR.

It is possible that that the annealing temperature might be too low. A temperature gradient may also help to identify the annealing temperature.

Increase annealing temperature and try DMSO in PCR mixture.

You can check the Tm of your primers using tools such as Netprimer, which is online and free. If possible increase then the annealing temperature accordingly (should be about 1.5 degrees Celsius higher than the Tm). Otherwise design new primers, which either have a higher Tm or target a different region of your gene of interest, where the sequence does not give you unspecific amplification.

Maybe, the primer were not spesific attach to the template from that isolate, so the bands were multiple. Or maybe the isolate still not single colony.

Try to check the Magnesium concentration, optimize the cDNA concentration and go for different annealing temperature by gradient PCR. Another u may need to reduce the primer concentration or check once running the primer sequence in blast

Check the quality of Taq polymerase that u r using for amplification along with Tm and other factors.

It may happen sometimes if your concentration of DNA is less. If more primers are available the chances are that they might bind at non specific sites. You can try increasing your template concentration, your annealing temperature or doing a MgCl2 titration. If none of these work, you can always depend on gel elution.

Thanks for the replies.

I'm performing it for 38 cycles. Length I don't know, if I'll get good results after amplification then i'll send it for sequencing. The result I'm expecting is around 376 bp.

Yeah I performed gradient PCR, in that i got the temperature i.e 48 C but if I'm Playing with this temperature. The thickness of the bands is getting low but still multiple bands problem is there

Its different isolate, on gel after DNA extraction I'm getting single band. After PCR only I'm getting multiple bands

I did it with positive and negative controls, the results are satisfactory.

i did check for that, there is no hybridization in primers. I checked it by loading positive and negative controls.

Its okay.

Its a genomic DNA. Only after getting the satisfactory results in PCR, I'll be sending it for sequencing.

I tried with gradient PCR also but conditions are same, only the density of bands got reduced.

I did gradient PCR,

Results in positive and negative controls are satisfactory.

On same day of extraction i'm performing the PCR

2 µl of each dNTP.

Along with it I tried it with Master Mix also.

No use i already tried.

I will try DMSO and let you know, no use of playing with annealing temperature, I already tried it

Yeah we placed the orders for new primers. For Tm, I took average of forward and reverse primers (+/-5) but its of no use.

The control was contamination free, and i tried it a couple of times. getting the same results

How to check the Mg concentration ??

I already played with different concentrations of primers, either the condition was same or I didn't get any results

You could try add BSA to your PCR mix (in our lab sometimes works) also you try do the touch down PCR, It helps in my few cases. If the bands are distinguish enough you can cut them all from the gel and send to sequencing to check what this band are. You can try this if you need only this PCR for sequencing not for some further procedure. If you get not very strong bands you can try re-amplification.

@Dharmesh Jain

Do not change all the parameters at the same time. Change one parameter at a time and check your band. To get rid of the non specific bands, increase your annealing temperature. What is your current annealing temperature?

I did not read all those answers above, so sorry if some one else have already said this: check your primer sequence on "primer blast" on ncbi. It is fantastic, as it will test whether these two sequences may amplify sequences on sense and antisense ref sequences; will test forward-forward and reverse-reverse combinations and the most important: will test few mismatchs between your primer and DNA, that will amplify in the real world... (especially mismatchs on 5'...)

I bet with you 5 million dollars that your sequences will show multiple targets on primer blast.

To solve this i would redesign either forward or reverse or both primers (is cheaper to redesign primers than struggle to optimize a reaction that your colleagues may not be able to reproduce afterwards...)

If I understand correctly it isn't problem with primers but with particular DNA sample, because for other isolate the PCR works just fine. So if you have some material left , maybe try isolate again.

I think that the problem is in the isolate, I agree with Magdalena Stolarek in here opinion , if you could repeat extraction of this isolate , best luck

Sorry, i read the question hurriedly, indeed may not be a primer problem. I agree the may be template (contamined with both DNA or proteins or degraded).. I can't help in any way but guessing...

If there is something with only one out of several isolates, then noting is bad with PCR conditions (primers, concentrations, Tm, etc.). How many extra band do you get? If one or two, then that could be the result of some duplication.

Dear Jain,

Try followings in the right order if you want to get single and clear bands and to not to pay much for PCR experiments:

1- Check your primer pair's sequence specificity for being sure that they don't stick anywhere except your target sequence. NCBI/Blast might be useful..

2- Change your Taq polymerase and try "monoclonal Ab-conjugated hot start taq polymerase and use its own "10x buffer" and do what "technical bulletin" says.

3- If it doesn't work, please try to change MgCl2 concentration gradiently; for example, try 1 mM, 1.5 mM, 2 mM and 2.5 mM MgCl2, but keep in limits, don't try 3 mM and more..

4- In another experiment, try to increase the amount of template DNA.

Have a nice experiment :)

Hi, As it is already specified by several scientists, I agree with that, it could be sample related problem. Your other isolates are working fine. Only you are getting trouble with a specific isolates. I do not know which kind of isolate you are using, but if possible please prepare a fresh isolate. best of luck.

It is a possibility that you primers are binding to multiple regions in your gene. The reasons may be that the primer sequence may be complimentary to certain regions in your nucleotide sequence. You may perform a PCR and try to identify the putative true clone with the desired size of amplicon. Also, a control reaction may be performed with single primer to identify the false amplicons.

You may also try manipulations in PCR components and conditions. Try increasing your annealing temperature

Hi there!

All answers above are realy good!

So just to add a bit here, I would certainly attempt to isolate the desired DNA sample once again. One reason maybe that the DNA quality is low as many others here have correctly pointed out to you. If the problem persists then redesign your primers as there maybe some sort of problem with the annealing. When changing conditions in a PCR, do change them one by one and not together otherwise you will get confused.

Concerning your target DNA one option is to sequence different parts of the DNA from which you want to amplify. If there is some sort of "repeat" of your target sequence then that may be a problem as you may be getting multiple bands since your primer(s) may be annealing at different sites.

Finally watch out for DNA degradation in cae the tube that you store your DNA may be containing DNAses. It's a pain and it could be a real problem.

Hope i helped!

Good Luck,

Nikos

Thanks for all the replies and wonderful info.

Okay will try BSA and the touch down approach

1.0.2 mM dNTPs (2 µL) for 20 µL reaction.

2,3,4. I'll try.

Yeah I'm changing only single parameter at a time

will do the blast and let you know,

yeah we redesigned new primers and placed an order

I tried it a couple of times

I already did that

yeah but i tried it also, and the primers are for the specific sequences so i think contamination shouldn't be a problem

I am getting three bands one is of high molecular wt. the other is near to my gene of interest and the third is of high molecular wt.

1,,2,3 I'll try,

4. I did but the band I expected shifted from ~376 bp to ~1200 bp around, product got amplified but still we are confused with the results.

I tried it a couple of times

i didn't do it with single primer will try it.

Yeah but when I increase the annealing temperature the density of bands got reduces, but multiple bands were there

We are taking proper care while isolating.

And the water we are using is DNAse free.

By the help for gradient PCR only we got to know the correct annealing temperature.

Annealing temp. we got

that I'll do by the help of blast

That I'll check

length is not an issue.

Anyways we placed order for new set of primers, will try it with new as well as old primers with little modifications in the protocols and procedures.

Multiple bands were found due to the non specific amplification, which could be avoid by using 5% to 10% DMSO. Although there are so many reasons to get Multiple bands. And more important is need to check Annealing temp..By changing annealing temp you might have get what you expected result.

DMSO goes and binds to the Cytosine part in DNA, it just plays a role in reducing the annealing temperature.

Solutions:

1. Re-design your primers (choose forward primer sequence started with C or G)

2. Increase annealing temperature

3. 3-5% DMSO

Hi.

Did you try to analysize the primer sequence in oligo software for primer design.

It may be about your primer,do blast for your primers and template.decrese anealing temprature then compare result.

Mohit Kumar are you sure its 1210 Kb?? what you wanna amplify. I had a good laugh!

Kindly go throgh the link if it helps. Some people have used different volumes of formamide also in the PCR reaction and have got good results.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC332902/pdf/nar00208-0251.pdf

I too getting same thing 3 bands nearby at required BP length one band  being thick and dark and 2nd and 3rd being little faint compared to dark one. Annealing temp is at maximum compared to other set of SSR primers which I m using for my study

Formamide is better than DMSO. It denaturates target sequences and primers. So increase specifity and yield of the PCR. You can use 1,25 - 5 % of formamide for the total volume of PCR. You may increase annealing temprature by gradient PCR, and use formamide.

Look also at this great file

Bhairavi Srinageshwar I am facing this problem for previously optimized reaction with very bright and specific bands, I have chosen the 62c annealing temperature, the highest in the row. now suddenly it gives me a non specific single band which is smaller in size, below 100. what could be the reasons. the primers were wonderful as I have checked them in silico also.

help plz