Biometra TRIO
Protocol Wizard
Version 03_19
65
10.4
Protocol Wizard parameter screen
For automatic calculation of PCR programs in the protocol wizard parameter screen
(see Figure 43) some parameters have to be set by the user:
1.
Touch the corresponding checkbox to select between two step and three step pro-
grams.
Note
: The protocol wizard offers to create two step or three step PCR programs. A
three step program consists of separated steps for denaturation, annealing and ex-
tension. In a two step program the annealing and extension step are combined to
one single step. In consequence for a three step protocol different temperatures for
the annealing and extension step are used and for a two step program the primer
annealing temperature T
a
(see below) is used for the annealing and extension step.
2.
Enter the PCR product length.
Note
: The product length is used by the software to calculate the elongation time.
The software uses the following equation:
Elongation time [s] = product length [bases]/1000 [bases]*60s For very short
products the minimum elongation time is set to 6s.
3.
Enter the primer annealing temperature
Note
: In general the annealing temperature (T
a
) should be set approximately 5°C
below the calculated mean primer pair melting temperature (T
m
). The theoretical
T
a
- value can also be calculated by the Biometra TRIO protocol wizard (see chapter
"Primer Annealing Temperature Calculator" p. 66).
4.
Enter the number of cycles.
To set the parameters touch the corresponding field in the protocol wizard parameter
screen and enter the desired values.
Figure 43 Biometra TRIO protocol wizard parameter screen
After setting all program parameters press:
N
EXT