Dear Yohannes

In general, within- and between primers complementary triplets increase dimers production. I would control it during primer design.

Also, I would not use primers with so different Tm (>5C).

Serial dilution of primers also a good advice.

Even if your primers are good, you still may not have amplification of your gene, due to biological or other technical reasons.

Check the OD260/280 ratio so you know the quality and quantity of dna. Get a definitely positive dna sample as a control to make sure the pcr can work and run it in all pcr reactions.

Use a hot start enzyme if you have one and if not then titrate the primers getting more dilute than you are using. try 1/2.1/4, 1/8 and 1/16 the amount you are using.

Run a temperature gradient of your annealing temperature from 10c below the expected annealing temperature to 5c above Ta

Add more magnesium in case your dna has pcr inhibitors and then start varying conditions once you have a dirty but positive amplimer to clean up the reaction

You have many choices...an easy one is to add some DMSO or BSA. Increasing annealing temperature a couple of degrees or in a gradient may also work.

Good luck

Roberto

To avoid primer dimer, here are some possibilities:

1) make sure that the concentration of your primer is optimized

2) The annealing temperature is optimised

3) The primer should be very specific to you target sequence

4) the PCR kits or supplier should be the good quality

Yohannes Tekle Asfaw From Qiagen:

https://www.qiagen.com/us/resources/faq?id=95f575e1-b477-4f97-a58c-922c69169c34&lang=en

How can I avoid primer-dimer formation during PCR amplification?

FAQ ID -544

Prerequisites for avoiding primer-dimer formation during PCR include the design of optimal primer pairs, and the use of appropriate primer concentrations. Complementarity of two or three bases at the 3' ends of primer pairs and complementary sequences within a primer sequence and between the primer pair should be avoided. Reduce the primer concentration to the lowest amount at which product amplification can be achieved by conducting test runs with primer concentration gradients.

Yohannes Tekle Asfaw I saw you have 3 primers listed. You can use online tools to check your primers. I like to use IDT company's primer analyzer, OligoAnalyzer (see below). Other companies also provides similar tools. Next time, you can check your primers first before you order them. You should also check other reasons why your PCR did not work. Did you include an internal control or positive control?

OligoAnalyzer checks primers:

✔ Length

✔ GC content

✔ Melting temperature (Tm)

✔ Molecular weight

✔ Secondary structure

✔ Self-dimer & hetero-dimer tendencies

✔ NCBI Blast

IDT: https://www.idtdna.com/pages/tools/oligoanalyzer

Increase annealing temperature and dilute your primers , Just use 10 Micromolar

Check with different anneling temperatures and decrease the concentration of primers.

You have to check first the conc. and the quality of your DNA sample as it may be degraded, the optimum annealing temperature for the primers and then run titration of the primers to get the optimum primer pair ratio suitable to give you good PCR products. You can refer to the following link for the primer optimization protocol:

https://www.sigmaaldrich.com/technical-documents/protocols/biology/primer-concentration-optimization.html

Try to optimizer your assay using a gradient PCR..

Also diluting your primer concentrations as suggested by Amy Johnson.

Goodluck

Try to optimize your assay using a gradient PCR..

Also diluting your primer concentrations as suggested by Amy Johnson.

Hi Yohannes Tekle Asfaw ,

It's so weird that does not exist a bit of the target but only dimer primer signal in your result..... I agree with Ruslan Kalendar only have self-dimers on your C. jejuni primer....

I guess than You have checked "in silico" all characteristics of your primers before use, and looking for interactions on itself and between them thoroughly . This is the most important step, You know....

I wonder if you use the 3 primers at the same time.... Maybe you should do ARMS-PCR in this kind of experiment. Anyway, You should use an internal control to check that your PCR has worked correctly.

I agree with the other colleagues. If the design of primers is correct, the Ta and primers concentration must be checking empirically doing gradient temperature, probing different primer concentrations, dilutions,...

Good luck!

Hi, Yohannes Tekle Asfaw

In order to help you a little bit more, I'd like to have afew answers about your experiment:

1) Do you have prior experience doing PCR?

2) Are you doing an end-point PCR. Right?

3) I do believe you did a DNA extraction and, at the end, analyzed the DNA concentration. If so, What was the yield on your samples? How much did you apply on your PCR reaction?

3) Please, let us know your PCR protocol (Concentration, not volumes, of reagents. Ok?) and cycling conditions (Enzyme activation temp - If it is a hot start enzyme - denaturing, annealing and extension temps and time, if you performed a three-step protocol).

4) If you did a gel electrophoresis reading, please, send us an image ...

I am waiting your answers in order to give you a better advice.

All the best.

Hi,

Using temperature gradient method, mean adjust annealing temp.

Check concentration of primers(better to decrease the concentration to avoid primer-dimers), magnesium chloride and temperature of the reaction

Hi,

you could also try to add some additives like DMSO or Betaine and see if that might help.

As others have suggested, you can try gradient PCR (varying annealing temperatures), dilution of primers (make a fresh dilution of primers) or the addition of DMSO. You need to try one by one to see which one works for you. Also, it is kinda too late but always make sure to check the potential self-annealing sites, potential hairpin formation, etc. using some online tool before ordering the primers. I use OligoCalc (http://biotools.nubic.northwestern.edu/OligoCalc.html) for this purpose.

I agree Rajat Kumar Jha's suggestion.

Change anneling temperature and change concentration of primers.

Hi, there, below steps might be followed:

I agree with dulmi, change these conditions

Hello! The main cause of primer dimer formation is ineffective PCR.

To avoid primer dimer formation: 1) Primers should be tested for complementarity to each other and self-complementarity. 2) All components of the PCR mixture must be in sufficient quantity or in exсess, including DNA, for the PCR to be effective. 3) Annealing temperature should be optimal (not too low). 4) DNA and entire PCR mix must be free of inhibitors. 5) Primers must be effective in PCR and be optimal in lenght and GC composition. In this case primer dimer formation in PCR is minimal and additives are not necessary.

1.Avoid complementary clamps on the 3' end of your primers, e.g. GC or GCGC, etc.

2. Measure and adjust your primer and template concentrations to recommended values.

3. Use touchdown PCR (temperature gradient) to take guesswork out of annealing temperatures.

4. Use high-fidelity DNA polymerases, e.g. Pfu.