It seems to me that you have quite a lot of non-specific bands. Before doing anything complicated or expensive, I would repeat the experiment using various negative controls : i) both primers but non Template DNA, ii) only the 3' primer on the Template, iii) only the 5' primer on the Template. While this is running, check your calculations and primers design. If primers and calculations are OK, then proceed to improve specificity using the various suggestions proposed here, like hot start polymerase, Mg++ concentrations, temperature, additives ... Actually this will (hopefully) help you reduce non-specific bands but not tell you why they appeared in the first place.

good success !

You have a great many number of bands on your gel. Are you sure those primers are specific enough for your purpose?

Also, how do you know all members of your community have a nirK gene of the same length of 500bp? Can you do southern blotting to verify nirK genes in those bands?

Http://www.bio-rad.com/gexp/html/support/amp_central/ac202b.html

I assume by 'Tm' you mean the annealing temperature and not the melting temperature. There are countless reasons why you are getting a non-specific band, which is difficult to point out without knowing what exactly you are trying to do. But, I would start by checking your primers itself, did you design those primers? Have you tried checking specificity to your gene using programs like BLAST? Another reason could be high GC content in primer, which given your high annealing temperature, I think is a fact. Primers with such high annealing temperature generally tend to have non-specific binding. Try designing a new primer set. Other options would be to try altering the MgCl2 levels in your PCR mix, or trying a high fidelity polymerase. Finally, if the non-specific band does not bother your experimental results or any further processing of your sample, then I wouldn't worry about it too much, but like I said earlier, it is difficult to pin-point the exact cause without knowing further details.

You may try to do a gradient of temperature with your primers and diffents magnesium concentrations as well

… What about some more base on your primer?

Esle, the usual cooking stuff such as change stringency etc…

It is also depends on polymerase which you using. Be sure that you are using the hot start polymerase or chemically modified 10 x buffer.

Haven't you got a negative control?

Since you have lots of non-specific bands you need to work out toward increase specificity of primer annealing (assuming you design the primers and calculated the fragment size correctly).

Try using 1M betaine (final concentration in PCR mix) or 8% DMSO. Also, doing temperature gradient is a good suggestion and you can do it plus/minus betaine. Playing with Mg2+ concentration can be another option to increase specificity. Try 0.8 -1.5 mM range.

You need to have a negative control on your gel. Also try doing a titration of MgCl2 at different concentrations or adjusting the temperature. In addition you should check primer specificity in BLAST.

Try make your PCR using lipids drops. So, you can try to make a nested PCR. First, you must to use a set of primers to amplify a big fragment. After this, you make a second round with a internal set primers. This improve your specificity.

Check the following

1. Primer Specifcity,

2. Use of Hot Start Taq Polymerase or Try setting up the reaction in ice

3. Try Different Mg concentrations

4. PCR additives - TMAC, DMSO

What about the quality of your template?

you need to do gradient PCR with different temperature. It will tell the perfect tm for the primer. if after that you have same then need to deign another one. .

You could try reducing your number of cycles to clamp down on non-specific amplification.

You could try a nested-PCR, using an annealing time of 30'' or less. Then purify your product.

I would say go with [1] the Swati Ghosh's solution first for checking the right Primer Annealing temps.

Then go in the [2], [3] , [4] order as suggested by Ajith Kumar if that does not helps.

[5] The final extension step Temp. depends on polymerase for most cases it is 72C 5-10min

and also the chain reaction extension step is usually 72C with the extension time depending on polymerase efficiency [6] (so can be 30sec/kb or 1-2min/kb). What is polymerase did you use?

If nothing works still then there are two ways to approach:

[1] gel extraction of your desired product --> PCR using that as template

[2] Design nested sets of primers. One pair just outside your region of interest then followed up with your current pair of primers.

It seems to me that you have quite a lot of non-specific bands. Before doing anything complicated or expensive, I would repeat the experiment using various negative controls : i) both primers but non Template DNA, ii) only the 3' primer on the Template, iii) only the 5' primer on the Template. While this is running, check your calculations and primers design. If primers and calculations are OK, then proceed to improve specificity using the various suggestions proposed here, like hot start polymerase, Mg++ concentrations, temperature, additives ... Actually this will (hopefully) help you reduce non-specific bands but not tell you why they appeared in the first place.

good success !

First start checking the quality of your DNA preparation and primer specificity, because I also see different intensities among the bands below 100bp (either dimers or non-specific binding or unused primers) compared to the amplification of other non-specific products. Play with primer concentration and also try to do a gradient PCR reaction to optimize the temperature.

Also, consider Ajith and Bernard's suggestions! Good luck!

There could be many areas from where chances of non specific band increases as: 1.Causes Related to Cycling Times and Temperatures

a. Too many cycles were used : Use fewer cycles when template concentration is high, and use more cycles when template concentration is low.

b. Extension time was too long : Excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1 min/kb.

c. Annealing time was too long: Excessive annealing time may increase spurious priming. Use an annealing time of 30 sec.

d. Annealing temperature was too low: If the annealing temperature is too low, primers may bind nonspecifically to the template. The rule of thumb is to use an annealing temperature that is 5°C lower than the Tm of the primer. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature. For greater accuracy, optimize the annealing temperature by using a thermal gradient.

e.Thermal cycler ramping speed is too slow: If the ramp speed of the cycler is too slow, spurious annealing may occur due to lower temperature and sufficient time for nonspecific binding. If ramping speed is not set at the maximum speed for the cycler, increase to maximum ramp rate.

f. Calculated primer Tm was inaccurate: If the primer concentration is calculated incorrectly, the calculated annealing temperature will also be incorrect. To calculate the primer Tm, use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions). Use the lowest primer Tm when calculating the annealing temperature.

2. Causes Related to PCR Components

a.Primers contain impurities: Contaminants in primers may inhibit PCR. Use desalted primers or more highly purified primers. You can try to dilute the primers to determine if inhibitory effects exist, but do not add less than 0.02 μM of each primer.

b.Too much primer was added: Using a high concentration of primers may increase the chance of primers binding to nonspecific sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.

c. Primers were designed or synthesized incorrectly by user or manufacturer: Verify that primers have the correct sequence and are complementary to the template. Use a primer design program to avoid repetitive sequences, regions with high complementarity, etc. Perform a BLAST search to avoid primers that could amplify pseudogenes or that might prime unintended regions. Use the tool at www.basic.northwestern.edu/biotools/oligocalc.html with the default salt concentration and 0.2–1 μM primer (depending on your reaction conditions) to calculate Tm. Use the lowest Tm of the primers.

d. Impure dNTPs were used: Contaminants in the dNTP mix can lead to incomplete or incorrect amplification or PCR inhibition. Use high-quality dNTPs.

e. Too much Mg2+ was added: Using high concentrations of magnesium increases the likelihood of nonspecific primer binding and unwanted product formation. Reduce the amount of magnesium in the final reaction.

f. Impure water was used: Water could have been contaminated during prior pipetting events. Use fresh nuclease-free water.

Regards,

Bivek

You are usinG genomic DNA, is it right? i recommend you to check your primers if they amplify anotherwhere except your gene in the genomic DNA. use BLAST programme

1) Mg concentration and primer specificity. Look for possible primer dimers in your primer design

2) Normally only noticible with smaller products, the ratio of tracking dye to product can result in a faint smear that can be mistaken for another band.

3) primer contamination

Daniel wrote: "I am working on amplifying the nirK gene from whole community genomic DNA"

When you are targeting genes from a whole community of bacteria you should expect to find other targets being amplified. The % of genome copies that actually have your target gene may be very low.

Cut the band out of the gel and have it sequenced ... costs 30$

That will tell you if it is primer artifacts or a similar gene in a different member of the community.

It may even be that some members of the species that is your target genome have duplications of the nirK gene.

You could try nesting a third primer or using TAQMAN to reduce this.

The bands look common to amplicons amplified from community DNA sources. Since you have products, it is highly likely that the annealing temperature is not specific enough. And that your primer set targets multiple regions of the template(s). I: you could determine the annealing temperature by gradient PCR and always include a no template control to check on primer dimer.

Make sure to use clean mQ in your reaction. Use the elongation temperature suggested by the company (you use 68 C, usually this is around 72 C).

Goodluck,

TS.

Regarding the unespecific band, does nirK have pseudogenes? Maybe nirk gene from a different species in your sample. Blast your primers and check.

Use less cuantity of DNA

There could be many, many reasons, most of which have been outlined by people already. Start with the simplest changes:

- Gradient PCR to determine best Tm to use (on some single species gDNA if it's available)

- Dilute your sample gDNA to 1:10 and 1:100, even 1:1000 and try at the selected temperature

- Do a hot start PCR

- Add DMSO or another additive which will increase specificity

I would also follow the suggestion of doing a range of no template control PCRs, changing specific items each time - the primers, the water, the taq. Rule out contamination of any of these things before doing your head in trying a lot of various changes to your protocol. Speaking of no template controls, where is yours? You absolutely must have one in every single PCR, positive control helps too if it is possible. Good luck!

have you tried to sequence your unspecific band? it could help you to know if there is mispriming, or if it is a similar sequence, pseudogene, etc...

after making the right diagnosis, you can begin to solve the problem, higher Tm, longer or shorter extension times, etc.

good luck !

I think the reaseon of amplification of the unwanted band is that another homologous sequence exist in the template (genomic DNA). In that case, I recommend 3 ways to solve the problem.

1. Remake the primers longer length.

2. Perform a gel extraction.

3. Carry out a cloning (insertion to vector) and sequencing using that amplificants. And then select a wanted target.

It is better to redesign your primer especially the 3'-end as you get a community band which is not cleared by high annealing temp

I was getting a lot of unspecific bands when doing a PCR of a vector with the primers designed for the vector. I found that I was adding too much template to the PCR reaction and all was solved when I used less DNA. It's one alternative approach to try while troubleshooting.

I would suggest u to run off a gradient first... If that doesn't work out then, what i can think off is increase the extension temperature to 72 for 5min.... Hope this would help you

Have you checked your Taq optimum temperature? Isn't it probably 72? All comments are useful so if you tried them all and there was no result, better not to waste time and redesign... Sometimes it take less time to redesign than trying to optimize a primer... good luck... Share your success news finally. Let us learn from your experience...

Are you checking your primer specificity using Primer-BLAST after you design them?

Also make sure to avoid designing primers binding to repeat areas such as LTRs. The best way to do that is to use the in silico PCR of UCSC.

Please give me the DNA concentration, and the master mix composition. It appears that there are a lot of other non-specific sequences (including primer-dimers around and below 100bp), but not a trace of the specific one (at 500bp). Check the primer specificity (virtual PCR, primer-BLAST, Gene Runner...) , try lowering the DNA and primer conc. Also take into consideration possible contamination (apply freshly prepared aliquots of all reagents and change the Taq-pol). I think that further changes in the PCR program will not be of any help (except for a hot-start in case you haven't already used it).

As Bernard Couvreur says, I think it may be a problem of primer specificity. You always need to set some negative and positive controls. Maybe the positive is difficult to get if you don't have previous successful assays, but I would recommend you to check if your primers can bind somewhere else. I actually have not clear which one is the 500 bp band you look for, cause it seems that the bands you point with arrows are much thicker. Also try to check at your samples, giving different band thickness for your non-specific products (as far as I understand): does it make sense that you have more unspecific product in ones than others? or are all samples supposed to be equal?

So my advise: check the primers and if you don't find suspicious regions for annealing out of your region of interest, try to sequence the "non-specific" bands and see what's inside.

Good luck! :)

To me, you need design new primers if you are sure your template DNA is pure no contamination. If the whole genome of closely related species is available, BLAST the primers against it。I guess there will be multiple unspecified binding.

Check DNA concentration, try using less that might help. I also agree you should check your primers do a BLAST and see what else they might be recognizing. Consider re-designing your primers, I use a software called APE and Serial Cloner software to check the PCR Rxn.

Do a gradient PCR for the annealing temperature using higher and lower temperatures from that of the Tm. Usually is Tm-5, in your case you should include in your gradient 58C. Try using 72C as your extension temperature and make sure that you are using extension times of one minute per 1000 base pairs in each cycle, which in your case you are using 30sec.

Good Luck!

Also you should try to use 35 cycle there....maybe that will help :)

Primers may not be good in practice even if they are good in design.Common and advisable primer concentration range is 0.1-1uM each primer.if the primers are not prone to make the dimers, the high primer concentration would result in LADDER

Excessive amount of DNA can be lead to false priming and probably to poor DNA synthesis because of obstructed diffusion of long Taq polymerase molecules

Your primer sequence has to be more stringent. Similar sequence must be around your traget DNA. Hence that region is also amplifying simultaneously. Try to redesign your primers.

Secondly try to minimize the DNA concentration.

Beside previous advices from your frineds, which is really useful info, also I suggest to reduces the primer concentration.

Take a look in primer GC content and try to lower MgCl concentration in your mastermix

Have you checked whether your gene can be alternatively spliced? Some bioinformatic analysis of a target gene is always useful before the molecular biological stage.

I've seen this before when I was amplifying human papillomavirus DNA with consensus primers. We never got entirely to the bottom of it, but I suspect that it was caused by a region of high homology to the 3' end of one of the primers just upstream of the authentic site. In this case you might get a slippage reaction where the region between the 5' end complement and the 'false' 3' end complement loops out. I'd check the amplicon sequence for possible false primer targets, remembering that G:T basepairs are stable in PCR primers.

Suggestions made by previous contributors are valid. You could also have shared how you made the PCR mix reactions.Critical parameters include: Annealing temperature- perform gradient PCR, MgCl concentration, Primer- scale down you seem to be having dimers and template DNA- purity and concentration.

In addition, it will be intresting to cut the band , purify and sequence to see what it gives.

If all this doesn't work then, redesigning the primers will be the way to go.

Best wishes.

You have received some good responses here, but you could also try a 2-step (rather than a 3-step) PCR. With your high Tm's simply ramping between annealing and denaturation temperatures, passing through the extension temperature, is likely sufficient to amplify a 500bp fragment. Including a separate extension step only increases the possibility that mis-priming of the targets will generate spurious bands.

Have you tried a gradient PCR to optimise the annealing Tm? Also, I would use 72degC for the extension step

it seems there is some homology between your gene and another one .You should start with hot start. I also suggest to have 2 sets of primers ,one inside the first, which may bypass any homology.

Probably your primers are not specific. It seems that you haven't check them in silico. Another reason isthat the thermal profile of your reaction should be optimized (prepare temperature gradient form 55C - 65C). As I can see the temp 68C is used for elongation, why 68 not 72? What kind of reagents did you use for reaction?

Good luck

IF nothing helps, U can try "SLOWDOWN PCR" or "TOUCHDOWN PCR" for details google those. Sometimes it helps...

Non specific bands may appear, because the time in the annealing step is very long , You should try only 15-20 sec and TM -15, and then star to adjust the annealing temperature, i.e. by increasing 5°C,then 10° and so on.

I think, you can, increase the annealing temperature to more specialized the annealing phase.

I think you should increase extension temperature to 72,because we always use 68 to get a large fragment. Lower tempeture will lead to many nonspecific bands.

The problem could be that you may have some homology in the priming sites of your gene with another location in the genome. I think this is normal and you will not have any problem if you make changes in the primers structure (e.g. longer primers, different sites,...). I should also mention that some PCR techniques give random bands with smaller primers.

Article Comparative assessment of genetic diversity in some tomato c...

i used to amplify a lot of 16S rDNA of environmental samples earlier.

to overcome nonspecific bands we used to do "band stab pcr". simply resolve the pcr amplified reaction on a 0.8% agarose gel. take a sterile 26 gauge needle and stab 5-6 times on the desired band and then shake the needle in a fresh pcr tube containing all the pcr ingredients and then do pcr. in many cases it saved our day.

maybe this might be of some help to you

try a lower primer concentration. 0,3 - 1,0 pM.

Daniel Curtis, please keep us informed about your progress !!

If no progress, you can tell us what you have done, and what happened... we are all learning :)

I would definitely try using negative controls before anything else and then start messing around with annealing temperatures and hot starts etc.

I suggest you check your positive and negative controls if they are ok. Try specificity by increasing the annealing time. In some cases annealing time helps to reduce non-specific binding. You can also specify bringing down the primer Tm by 5C.

I faced this problem before and realized that my primers are not well designed. Good luck

It could be wise to check the DNA concentrations of your primer solutions. If unequal amounts of primers are added in the PCR, one strand of the amplicon will accumulate faster than the other strand and will appear as an additionnal band of ssDNA on your gel. Another way to identify the problem is to perform a short treatment with Mung Bean nuclease after the PCR. If this treatment removes the unexpected signal, then the problem is the unpaired accumulation of amplicon strands due to unequal primer concentrations, or a too large difference in their Tm.

What do you mean with "whole community genomic DNA" ? If you have several distinct organisms in your sample, perhaps it is possible to amplify a nirK variant with a deleted part. (I know nothing about this gene)

It could also be an artefact of the PCR if you have some repeat in the amplified region.

If not, to avoid unspecific priming, do you prepare your reaction in ice or add the polymerase after the initial denaturation ? Or do you use a "hot start" DNA polymerase ?

good luck

Probably it is impure DNA or extra primer.

Hi, Daniel Curtis

I think your primers were Improperly design , but it is better to use Touch Down PCR, this program is very useful to remove non-specific bands. In addition, it is better to reduce Mg concentration and you set annealing time 15 sec. in stead of 45 sec.

Hello Daniel,

Check the sequence of your primers that is written on the tubes with the original sequence you have. Blast them to see how specific they are. Have you designed the primers yourself or you have taken them from a paper? If it is ok, then run a gradient pcr to look for a perfect annealing temp.

I always blast the primer sequence to check for its specificity before ordering them.

Try this. Wish you all the best. Keep us informed with the development.

I would just gel extract and purify your band of interest, you might be having problems with non-specific binding of primers

Non specific amplification will be reduced by use of Thermostable MutS (and RecA).

Please read the following papers.

(1) Fukui, K. et al. (2013) “Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during PCR”, Int. J. Mol. Sci. 14, 6436-6453 (open access)

http://www.mdpi.com/1422-0067/14/3/6436

(2) Fukui, K. and Kuramitsu, S. (2013) “Simultaneous Use of MutS and RecA for Suppression of Nonspecific Amplificatioin during PCR”, J. Nucleic Acids 2013, ID 823730 (open access)

http://www.hindawi.com/journals/jna/2013/823730/

The plasmids for protein production of Thermus thermophilus HB8 are distributed from RIKEN Bioresouce Center. They have constructed the sophisticated system for distributing our resources concerning T. thermophilus HB8.

The plasmids for MutS and RecA are also included.

---------------------------------------------------------------

1. Plasmids for Overproduction of Proteins from Thermus thermophilus HB8

We have already deposited a plasmid set which includes more than 2,000 clones of Thermus thermophilus HB8. This plasmid set can be obtained from RIKEN Bioresource Center (BRC).

http://dna.brc.riken.jp/en/thermus_en.html

MutS (TTHA1324) and RecA (TTHA1818) are included in that plasmid set. If you will use several clones in the near future, I recommend you to ask a set of plasmid DNA (not transformant of E. coli) to RIKEN Bioresource Center. And please distribute them to anybody who wants.

2. Plasmids for Gene Disruption

If you want to analyze the role of a protein (or gene), the gene disruptant of T. thermophilus HB8 can be obtained from the gene disruption plasmid. About 1,000 plasmids for disruption of the T. thermophilus genes are also distributed from RIKEN Bioresource Center.

http://dna.brc.riken.jp/en/thermus_4en.html

---------------------------------------------------------------

If there is any trouble, please don't hesitate to contact me.

I hope your success in your research.

1/ Your primer Tm is 63C you should 2 degree increase or degrees your Tm... and use Touchdown 68-58 PCR

2/ may be your Template DNA is higher then the required quantification use 30 ng/µl DNA

There could be two explanations for your problem:

1) Technical problem with your PCR. Solution: try the common ways to solve unspecific banding (temperature increase during primer annealing, go even higher than 66°C or increase gradually MgCl2 concentration of your PCR buffer, decrease template amount, decrease primer amount or decrease enzyme concentration. If nothing helps you may want to add adjuvants like BSA or DMSO)

2) Not every nirK gene of a pool of denitrifying bacteria has the same length. So you may very well have in reality different amplicon sizes, meaning that different bacterial species possesing nirK are present in your total DNA pool. Clone the gene in for instance TopoA cloning, selct positive E. coli clones and sequence some of the positive clones. A blast can tell you if you have different species in your community

HI Daniel, 

I agree with most of what has been posted. It seems your product is GC rich. I would suggest using a GC rich PCR buffer and enzyme. I think Takara or Qiagen have these products. Have you blasted the region your amplifying ? My advice would be  design primers to make a larger PCR product and  see if you get amplication and if so whether you get the secondary band. The other thing it could be is, secondary structure forming some sort of heteroduplex.