Hi,

I wonder whether there is any problem with your template. Are you sure that you have enough DNA amount in your template sample?

What final concentration of dNTPs and Taq was used?

You may use a fresh set of buffer, dNTPS and Taq enzyme in order to make sure that there is no issues associated with assay components.

You supposed to be curious with your oligos. Please upload your oligo details. Your results blindly suggest us possibilities of non-binding to the target region. You better have the Tm of oligos relatively higher as 62-64'C; which implies a relatively lengthy oligos in your assay.

Specificity could be increased by ‘hot-start’ approach by adding the Taq Pol after your initial denaturation step.

A gradient PCR would help in choosing the best annealing temperature for your assay (This is applicable when you get a product).

You may avoid unnecessary non-specific annealing by performing your assay preparation on ice and plan your assay with appropriate concentrations of the components.

However, the important factors to play with are the Tm of your oligo while designing it and the annealing T.

Good luck.

do you also get your PCR product too, if so, then why worry about primer dimers, let them be their as far as you are only concern about the PCR product... if not then your primers are not well disighned to do the job, design new sets of primers, probably use as less as possible.

No I am not getting the PCR products.

Hi,

I wonder whether there is any problem with your template. Are you sure that you have enough DNA amount in your template sample?

What final concentration of dNTPs and Taq was used?

You may use a fresh set of buffer, dNTPS and Taq enzyme in order to make sure that there is no issues associated with assay components.

You supposed to be curious with your oligos. Please upload your oligo details. Your results blindly suggest us possibilities of non-binding to the target region. You better have the Tm of oligos relatively higher as 62-64'C; which implies a relatively lengthy oligos in your assay.

Specificity could be increased by ‘hot-start’ approach by adding the Taq Pol after your initial denaturation step.

A gradient PCR would help in choosing the best annealing temperature for your assay (This is applicable when you get a product).

You may avoid unnecessary non-specific annealing by performing your assay preparation on ice and plan your assay with appropriate concentrations of the components.

However, the important factors to play with are the Tm of your oligo while designing it and the annealing T.

Good luck.

change the primers if you are getting only primer dimers and not the PCR products. I think this is the only solution and even changing the temperature may not result in proper product formation. Anyway please inform about the solution you used when your problem gets sorted out.

I also think you should redesign your primers. There is a lot of help in the web for primer design and avoiding of primer dimers.

But as you do not get any PCR product, Navaneethaiyer Umasuthan made an important point; make sure that all components are fresh and useable (especially the dNTPs), and not contaminated.

Good luck!

I think it's mainly because of the concentrations of components (if you're sure about the primers and purity of the components). please note that Mg ion, for example, is a cofactor for Taq polymerase. So if the concentration of Mg ion be insufficient, the Taq polymerase cannot proliferate your desired region, even if your components be fresh.

Dilute the primer concentration.

Change the concentration of the primers for lower (I usually us 5-10 pM), try add Mg2+ to the PCR mix or add more DNA template. if any of that not working just design new primers

You should also check your PCR conditions - how big is your fragment, is the elongation time long? Have you checked the annealing temp? And are you shure that the primers bind? Do you have a positive-controle you could use?

You can try using 1% BSA or 1-2mM TMAC in your PCR mix long with buffer, dNTPs, primers, Taq, and 40 - 100ng DNA. It will work.............Good luck

Pls maintain the temperature and avoid more AT pairs in your primer. Do it in white light

Hi, sometimes there may be a problem with the amplicon GC% - if it's too high, you may get nothing even if the primers are fine... try adding 5% final v/v of glycerol or DMSO to the PCR mix, and see what you'll get. There is a variety of other additives and enhancers that you may try, but these two usually work well. Good luck!

Can I make a suggestion? If you give more details about your PCR, you will get a much better answer. You are leaving too much to our imaginations and making us guess what could be the issue, but if we had all the information we could make educated guesses. :)

One thing I always to in my PCRing is run both a positive and negative control. The positive control being DNA and primer that has given me product in the past (lets me know that all my buffers and enzymes are okay), and the negative control which is everything BUT DNA (this will show me what is happening, hopefully nothing, if the primers have nothing else to do and if there is contamination in any of my other reagents).

Target Region: How long is it? GC content? Betaine also works to even GC to AT basepairing Tm.

Primer design: What are the sequences? What did you use to design primers? I like Primer3 (to get a list of possible primer pairs) and Perl Primer (to check for extendible primer dimers) and IDT Oligo Analyzer (to check for hairpins) (all free). What DNA sequence did you use to design your primers? Were there introns and exons, or did you use the exonic sequence only? If you are also trying to amplify introns across two exons, then you may be amplifying a very large fragment.

Buffers: What buffers are you using? Do you make your own (what is the recipe?) or do you have pre-made? I mostly use 10X Taq buffer or Epicentre (I am bias, and I get this cheap) 2X pre-mix E for most applications.

Thermal Cycling: What is your program? What is your annealing temperature compared to your melting temperature? What is your extension time? How many cycles?

Once we have this info, your answer will come so fast, and you will not have to test so many things.

I suggest that you redesign your primers and change them or adjust their concentrations..