Hello, i am trying to get a point mutation using overlap extension pcr.
The first round of pcr work excelent, i get my 2 bands, from external primer foward to mutagenic primer reverse, and from mutagenic foward to external reverse.
The problem is in the second one, the reaction just can't work properly!.
I can't get the third band between the two external primers, i only get the two bands mentioned before.
i tried with temperature ramp from annealing and magnesium ramp, but i get only the same two bands, the third band doesn't appeared!.
i tried to give more denaturation time in the pcr cycles, but when i did it, i don´t have any amplification (and here i have another question... if i give a lot of time of denaturation (i gave it 5 minutes), why reaction doesn't work? even the control doesn´t amplified ).
thermal cycling conditions : initial denaturation 10 minutes to 95 °C, denaturation 45 sec. to 95°C, annealing 60 sec. to 50°C, extension 60 sec. to 72°C, and a final extension 10 minutes to 72°C x 25 cycles
I just can't understand why it doesn't work, i use the same protocol in other point mutation, and it works great.
I pray for help
Thanks in advance
Edited by Guierdy Katherine, 10 January 2014 - 08:41 AM.