Primers will anneal at all temperatures below the temperature that they fail completely. As the annealing temperature drops the primer can anneal to an increasing number of similar complementary sequences in your genome, the pcr becomes less specific and multiple amplification bands appear. The gradient pcr is probably simply to find an annealing temperature that gives both good yield and clean amplification. The genomic sequence itself will also effect primer annealing as sequence changes under the primer will always have an effect on primer annealing

We used to calculate the annealing temperature of primers based on the A,G,C,T content. But it will be good to conduct a gradient PCR using a range of temperature in and around the calculated annealing temperature to know the temperature which gives maximum amplification. The same reagents with all the components in the required proportion should be used for the amplification of DNA as the concentration of components like MgCl2 is also important.

Hi, Muhammad

- Gradient temperature is usually the best way to get your best annealing temperature & conditions for your primer set, and it saves time!

- each primer set could have more than annealing temperature, and different primers sets may have the same annealing temperature, as well, and usually the best ones for each primer set is 3oC range (e.g. you primers may give you the best amplification results with the clearest band (amplicon) at 57oC, but it also will be good at 56 oC &58oC), all should be ok, consider it while setting your gradient conditions.

- Consider that the best annealing temperature usually (55oC-60oC), unless you have high GC content in your primers, so you should consider higher temp.

Good luck

Yes.must be same and more than or low than at 3-5 temperature

The annealing temperature should be only one.Sometimes the annealing temperature of the primers which are depicted in the paper do not match with the practical approach. In thar case primer standardization is required.

Dear Paul Rutland , Sonu S Nair , Eman M Taher , Hasan N. Smesam , Soumyajit Biswas ,

Thank you very much for your kind responses.

I understood your points of view and they are all correct in their place.

But I couldn't make myself clear in asking the question.

Letme explain with an example:

for example I use a primer set matk as universal primer on let say 100 plants.

out of 100 it gave positive results for 30 plants at annealing temperature of 48.

lets say it didn't give amplification for rest 70 plants, so should I run gradient for all of them or use the temperature 48 as standard for all the future plants, will 48 always give amplification? or exceptional cases are there that it might give amplicons at different annealing temperatures?

I hope I make myself clear this time.

Thanks in advance.

I think you should standarize each of the sample to find the actual annealing temperature.From my practical knowledge I can tell that the annealing temperature varies according to the laboratory conditions and sample type.

In the example that you gave of 100 plants, I would be more concerned about PCR inhibitors than I would be about the annealing temperature of the primer.

Dear @kevin,

I do face problems with pcr inhibitors mostly as I extract DNA from herbs which contain alot of impurities.

What do you suggest in that case would be solution of my problem?

Regards

All samples should be standardized and select one annealing temperature for all