Effect of Temperature on PCR - (Sep/22/2006 )

I have been trying to standardize the conditions of my RAPD PCRs. I noticed a strange thing. Well its strange to me cause i dont know the reason but the first papers on RAPD also indicate its authenticity. I noticed that even if the calculated melting temperatures of my decamer primers was round about 32 C, I got better bands as i increased the temperature at the annealing step of PCR and the best bands were seen at around +5 to +6 degrees C of the calculated melting temperatures. Moreover when i set the annealing temperature at -3 to -4 degrees C of the calculated melting temperatures i got smears in between the bands. What is the reason of this????

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In general thats how PCR works .
By increasing the annealing temperature you're increasing the specificity of your primers to the target regions. while when temperature is decreased non-specific binding results in multiple bands

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Well I know that much but if increase the annealing temperature a few degrees higher i get no result at all i mean around +7 to +8 of the calculated melting temperature. Now +5 is also more than the melting temperature so shouldnt it make annealing difficult like +7 degrees C. Or is there some specific range. Another thing whereever i,ve read about setting temperatures of PCRs for designed primers they usually tell us to set the annealing temp around -5 C. e.g on the fermentas kits and taq packets and usually we get good results but not in the case of rapid. Now if the results are authentic for +5 C annealing temperatures in RAPD why are they not so for +7 C temperatures???

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How did you calculate the Tm? Unless you use the really anal calculations, it's always going to be an estimate.

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