how to amplify a 350bp in length from a vector - (Sep/06/2005 )
i have tried several times ,but still no band was amplified. I am confused now.By the way, as i add enzyme sites at the 5'and 3', so i divide the program into two parts:the annealing temperature of the first 5 cycles is 5 degrees lower than the next 30cycles. I have tried several annealing temperature,but all fail.
-yanshi-
Why would you want to increase the annealing temperature during PCR?
Have you tried different Mg-concentrations, how much is your input DNA, are you sure about the quality of your DNA, is your enzyme still ok (perform a control PCR on a different template)?
To make sure you get some product, go to a very low annealing temperature, say 10°C beneath the Tm of the primer with the lowest Tm.
-vairus-
QUOTE (vairus @ Sep 7 2005, 12:19 AM)
Why would you want to increase the annealing temperature during PCR?
Have you tried different Mg-concentrations, how much is your input DNA, are you sure about the quality of your DNA, is your enzyme still ok (perform a control PCR on a different template)?
To make sure you get some product, go to a very low annealing temperature, say 10°C beneath the Tm of the primer with the lowest Tm.
Have you tried different Mg-concentrations, how much is your input DNA, are you sure about the quality of your DNA, is your enzyme still ok (perform a control PCR on a different template)?
To make sure you get some product, go to a very low annealing temperature, say 10°C beneath the Tm of the primer with the lowest Tm.
hi，
thank you for your advices.i have tried again and amplified the band this time
-yanshi-