Question

Select an annealing temperature based on the Tm of the forward and reverse primers. Briefly justify your selection

Select an extension time based on the expected length of the amplified DNA fragments. Briefly justify your selection

Answer 1

Annealing temperature is the temperature at which the primers recombine to the template . It depends on the base composition. Normally it is kept around 3-5 degrees less than the Tm of primers . It should be kept as high as possible so that specific annealing can be done. Not higher than the melting temperature of any of the primers as in that case the primers will start to break. So it is kept close to the Tm of primers. Keeping a very low annealing temperature leads to annealing of one or both primers in sequence other than the desired result.

Extension of DNA fragments can be done at a temperature of 68 degree Celsius. The time depends on expected length of fragment.

Extension time of 1 minute is needed per 1000 bade pair or 1 kbp. For less than 1 kbp, extension time of 45-60 seconds, for more than 3 kbp, longer extension time may be needed. Sufficient time should be given for the annealed fragments to extend.