Biometra TProfessional Basic Gradient - Page 48

46 Instruction Manual TProfessional Basic 06/2011
Dwell 15sec, Annealing temperature 60 °C
95 °C 5:00
96 °C 00:15
59 °C 00:15 35x
68 °C 00:30
72 °C 01:00
16 °C Pause
Dwell 10sec, Annealing temperature 60 °C
95 °C 5:00
96,5 °C 00:10
58,5 °C 00:10 35x
68 °C 00:30
72 °C 01:00
16 °C Pause
Dwell 5sec, Annealing temperature 60 °C
95 °C 5:00
97,5 °C 00:05
57,5 °C 00:05 35x
68 °C 00:30
72 °C 01:00
16 °C Pause
Dwell 1sec, Annealing temperature 60 °C
95 °C 5:00
99 °C 00:01
56 °C 00:01 35x
68 °C 00:30
72 °C 01:00
16 °C Pause
The protocol templates are pre-installed in the directory “Biometra Shared” (see chapter 5.3.1).
Note: The given annealing temperatures correspond to a calculated primer annealing
temperature of 60°C. If necessary, the annealing temperature has to be optimised for each
single primer pair. Biometra recommends to optimize the annealing temperature by a gradient
at 0.5 °C steps on a gradient enabled PCR Thermocycler.
Note: For the protocol templates an elongation time of 30 sec each is given. For optimisation of
the elongation time please take the processivity (bases/sec) of the Fast-PCR polymerase given
in the supplier handbook into consideration and adjust the elongation time corresponding to the
length of the fragment to be amplified. At a processivity of 35 bp/sec the elongation time given
in the protocol templates would be sufficient for the amplification of a 1000 bp long DNA
fragment.
Note: The proposed protocols are only applicable for the Thermocyler TProfessional Basic 96.
To establish a Fast-PCR at the Thermocyclers TProfessional 96 and TProfessional Standard 96
please follow the instructions in the homologue chapter of the corresponding manual.