Hi Krisztina,

Great question. I always do a annealing temperature gradient whenever I get some new primers, starting from 50-70deg in a 8 well strip. Don't even bother really looking at the predicted Tm beforehand, as it will change depending on your enzyme, buffers, PCR machine etc. Once you have done the PCR, simple run it on a gel. What you are looking for is the highest temperature you still get a good product, ie. good band on the gel, after which the temperature is too high and you lose product, i.e.. the band disappears. Hopefully, you will only see a single band at the expected size! If further Tm refinement is needed, you can have a smaller gradient range on the next PCR, however, this is usually not necessary.

Does that make sense?

Dear Stephen, thank you! I have just read your first publication on this ResearchGate, to know you specific area. As a beginner we are intend to try out a basic method like RAPD, and in this case I expect several bands using decamers as primers. But anyway, I hope that simple logic works in this case as well, so the highest temperature with the utmost clear good bands will be the optimum annealing temperature...? What I was still thinking on, is that how the annealing time can influence my results. :O)

I will try to find your publication as a full article, thanks to show up and showing the path for me!! Krisztina

No problem at all :)

If you are using a random primers and expect several bands, then it might be worth dropping the lower gradient temperature down to 45 and going up from there, just to see what the pattern looks like. This will allow for a greater range in annealing of the random primers. I probably wouldnt increase the Tm all the way until the PCR stops working, as you are likely to miss some polymorphism - just increase the temperature of the gradient until you see a suitable pattern of bands that can be distinguished form each other.

I am assuming you are working off genomic DNA? My experience has been that the dirtier the sample, the longer the annealing time. SO it depends on how you have purified your DNA. We typically use 30-60secs, which works fine. If its purified genomic DNA, you could go shorter.

Good luck, and let us know how you go!!!

Steve :)

Thanks! Promise to come back - leastwise with questions:O)

Thanks.

(So the door has just been opened, and I feel quite at home here on ResearchGate...)

this pdf would help you a bit....

Thanks All