start at a temperature which will be too hot and not work and work downwards to get maximum specificity for a couple of cycles on one primer before the other primer starts to work,Somewhere about 6c above the Tm of the highest TM primer should be ok for touch down pcr. For hot start use the correct annealing temperatures but either use a hot start enzyme or heat the mix without enzyme to approx. 80c then add the enzyme quickly and start cycling

start at a temperature which will be too hot and not work and work downwards to get maximum specificity for a couple of cycles on one primer before the other primer starts to work,Somewhere about 6c above the Tm of the highest TM primer should be ok for touch down pcr. For hot start use the correct annealing temperatures but either use a hot start enzyme or heat the mix without enzyme to approx. 80c then add the enzyme quickly and start cycling

• I basicallyagree with Paul.
• I would elaborate by saying that in touch down I would start at TM +3C and progress down to TM-3C in 0.5C intervals per cycle. Therefore 12 TD cycles
• before commencing PCR with a further 20 cycles @ Tm-3C in a standard 3 step PCR

I was working with TD-PCR for cloning a zebra fish protein and I used this method of PCR obtained very good result. For this method I used the Tm 3°C over the optimal Tm and configured termocycler and low the temperature 0.5 °C in each cycle

The hot start PCR approach simply make use of a polymerase that is inactivated at room temperature, e.g with an attached antibody that release at 95 deg C for 3-5 minutes to activate the enzyme. It makes it relatively safe to set up PCR reactions without chilling the reagents (to prevent polymerization at sub-optimal annealing temperatures that can lead to unspecific amplification). For touch down PCR I agree with the above.