How do I find a good annealing tp for a PCR cycle? Why the primers don't melt at the extention tp that is> Tm? - annealing tm of my primer
1 I know) that the annealing temperature should be lower due to the Tm of the primers in the Tm of the primer will melt, we have ignited us for a temperature of annealing below the Tm, so that the first model (TM, if choose) to 60 ° C, annealing temp must be 55 to 59. Ok. Thant), but then the temperature at 72 C, by extension, why the caps do not melt at this temperature (which is no longer their TM.
2) in the laboratory, in real time using primers SIBER Green C 45, MC and the cycle of the annealing temperature is 55 ° C are used and have good result, "How?" If I can help someone would understand it greatfull thank you
Sandra
Wednesday, January 27, 2010
Annealing Tm Of My Primer How Do I Find A Good Annealing Tp For A PCR Cycle? Why The Primers Don't Melt At The Extention Tp That Is> Tm?
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Hydrogen-1) between A & T and C & G hold together the two strands. Thus, Tm increases with the duration of the two channels. Should therefore be up to 94 t in the DS-denatured DNA increased. Can not be 72 degrees is temporarily no longer, the better. We know that the polymerase works very quickly. My guess is that high enough by the time t to the first extension there is enough distance to son to stick together. Please let me know when you get to be an authoritarian.
2) TM than the point where half of the DNA double-strand distances and is defined in a single string. In half of the primer Tm not be combined. In addition, the melting temperature is not hard and fast numbers, is a good proposal for a thermodynamic equation is based. You need to play with the annealing temperature to optimize the reaction.
Lowering the annealing temperature tend to produce the product and increases the nonspecific amplification. The increased temperature provides a concrete answer. People who are in real timewant to avoid all non-specific, because they get rid of their results. Likely to fall at the same time increasing the annealing temperature, the amount of product in the wells will be checked, and know when you want to measure the amplification of the amplicon to see not a doppelganger to.
Enjoy!
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