The length of time could reflects the size of the product. Longer product needs longer extension time. Also if the annealing temperature is quite low, it will take longer for the machine to adjust its temperature from denaturation to annealing. Some of the older protocols use long extension times because back when the protocol was created many years ago, the DNA polymerases were very slow . We have much faster polymerases now, so it pays to optimise your protocol. You could try incrementally reducing extension times to see if you can cut down on the length of the protocol. With some of the newer polymerases, your extension time need only be 10 seconds or less if the product is less than 1000bp. Also, the shorter the extension time the less likely you are to get non-specific product.

Dear Endalew:

Total time required to complete PCR reaction most probably depending on a number of issues, such as: (1) number of total cycles, (2) the program steps for each cycle (i.e. time setting for denaturation, primer annealing, primer extension, final elongation) and (3) the PCR machine used in the PCR run.

(1) The more total cycles, the longer the time. If you use different number of total cycles among the three markers then you will end up with different total time required,

(2) The program step of each cycle, the longer the setting for denaturation, annealing, primer extension and final elongation - the longer the total time

(3) The machine, older generation of PCR machine need more time to change the temperature from one steps to another. Therefore, it will require more time to complete PCR reaction. The latest PCR machines most probably need less time.

This may not be the reason if you used the same PCR machine for your three PCR reactions.

Last  but not least, numbers of newer generation of PCR kits have been around which require less time for each cycle. Therefore, if you used this you may end up finishing the PCR reaction quite fast than what you used to.

However, this again did not answer your question if you used the same PCR kit for all three PCR reactions you did. In conclusion, my bet would be that point (1) and point (2) were the reasons for time differences.

Good luck...

Thank you for the above comments.

I agree with the previous answers, I think you need to check the actual conditions of your PCR, the length of time could reflects the size of the product.

Its true, if the annealing temperature is quite low, it will take longer for the machine to adjust its temperature from denaturation to annealing. And maybe, total time required to complete PCR reaction most probably depending on a number of  total cycles and the program steps for each cycle.