Hello,

Most of the comments above might be useful for regular PCR, but they are not completely applicable to RAPD. As 10mers, the primers should be able to bind by chance in almost any genome and produce at least some bands. You can increase or decrease temperature (usually not below 34 or above 40 C) if you are getting too many or just a few bands.

It is not advisable to standarize conditions for individual primers since usually for RAPD you have to work initially with a lot of primers and select out of them those with the highest number of bands so you will have more chances of finding polymorphisms in your samples. Besides, results from all primers used are combined for distance calculation, so it is always better to work similar conditions for all of them.

We have worked with different plant species and the same conditions work perfectly well for every case (just different primers give better results for each species). From the smear I see in your gel, it looks like some common effect we see when changing species: you might be using too much DNA. We have never used more than 20 ng in a 25 microliters reaction, even for species with big genomes. The smaller the genome, the lower concentration of DNA you would need, since you will have more potential binding sites for the primer per ng.

Before trying with different PCR conditions, try using 10, 20 and 50 ng and see if there are significant changes in your results.

Please check this publication for the PCR conditions we use:

https://www.researchgate.net/publication/40668760_In_Vitro_propagation_of_enterolobium_cyclocarpum_(guanacaste)_from_nodal_explants_of_axenic_seedlings?ev=prf_pub

Article In Vitro propagation of enterolobium cyclocarpum (guanacaste...

I tried a few weeks ago more than 5 different commercial RAPD primers. First I proceeded to gradient PCR. If you want I can send you the sequences and additional information (tm etc.) I think that the problem is on primers. Another difference with my protocol is that I used double concentration of primers (40uM), but I don't think that this is the problem. What temperature did you used? How about the other parts of the program?

We had at first the same problem with RAPD for fig DNA, we used gradient pcr for annealing temperature and also increased the elongation time, and number of cycles.

Good luck

Thank you all for suggestion,I guess I have to try with gradient PCR .

@ sumaiy Nabi i kept annealing as it was mentioned in primer certificate of analysis from the company where I ordered Primers.@Christian , Rana I tried with increase of elongation time I kept 40 cycles still the same problem .

Today i will try with the gradient PCR and also decrease of primer conc. as you all suggested hope I get some positive results

Reduce amount of the Taq 0.3 in 25 micro liter of the reaction. Standardized your primer using gridient PCR to find out the optimum temperature surely you will get success. All the best

Hello,

Most of the comments above might be useful for regular PCR, but they are not completely applicable to RAPD. As 10mers, the primers should be able to bind by chance in almost any genome and produce at least some bands. You can increase or decrease temperature (usually not below 34 or above 40 C) if you are getting too many or just a few bands.

It is not advisable to standarize conditions for individual primers since usually for RAPD you have to work initially with a lot of primers and select out of them those with the highest number of bands so you will have more chances of finding polymorphisms in your samples. Besides, results from all primers used are combined for distance calculation, so it is always better to work similar conditions for all of them.

We have worked with different plant species and the same conditions work perfectly well for every case (just different primers give better results for each species). From the smear I see in your gel, it looks like some common effect we see when changing species: you might be using too much DNA. We have never used more than 20 ng in a 25 microliters reaction, even for species with big genomes. The smaller the genome, the lower concentration of DNA you would need, since you will have more potential binding sites for the primer per ng.

Before trying with different PCR conditions, try using 10, 20 and 50 ng and see if there are significant changes in your results.

Please check this publication for the PCR conditions we use:

https://www.researchgate.net/publication/40668760_In_Vitro_propagation_of_enterolobium_cyclocarpum_(guanacaste)_from_nodal_explants_of_axenic_seedlings?ev=prf_pub

Article In Vitro propagation of enterolobium cyclocarpum (guanacaste...

I fully agree with Gustavo in the sence of temperature optimization. I have used 16 standard RAPD primers with annealing temperature of 36 C (which is usually the optimal t for RAPDs) and there were always a lot of products. Even in 35 or 40 C - similar results.

However, DNA excess was not a problem in my experiments, but instead it was DNA quality. The DNA must not be degraded by DNAse. If you want to find polymorphisms, make sure your DNA is compact (agarose electrophoresis, or some specific duble-strand DNA fluorimetric quantification, such as Qubit). And, contrary to Arun, I used 2 u recombinant Taq poly per 25 ul reaction.

Wish you all the luck.

Dear Pavan,

We standardized the RAPD reactions in our lab. Works for almost all RAPD primer and all the species we tested (most plants).

Mix: 5 ng DNA, 1X buffer, 1.5 mM MgCl2, 1 U Taq, 0.2 mM dNTP, 0.2 mg/ml BSA, 2.3 ng/uL primer (final volume: 13 ul).

40 cycles: 92°C 1 min, 35°C 1 min, 72°C 2 min. Final extension 72°C for 5 min.

Thank you all for suggestion, i am getting very useful results ... Tanks a lot

I also got the same results and my lab-mates told me that my mistakes were using forward primer and not using revised one. please advise

Dear Ammar,

my mistakes were due to some contamination in buffers , change ur annealing temp. go for gradient hope you ll get results .. good luck