Applied Biosystems GeneAmp PCR System 9700, Руководство пользователя

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Creating and Editing Methods 5-9
Defining Pre-PCR
Holds
The Hld field on the Create screen defines the number of holds for the pre-PCR
segment of your method. One (1) is the typical setting for most PCR amplifications
and is the default value for the Hld field.
Generally, pre-PCR holds define a temperature and hold-time sufficient to denature
any endonucleases or exonucleases that may contaminate your prepared samples.
Defining PCR
Parameters
The Tmp field on the Create screen defines the number of temperature control
parameters in the PCR cycling segment of your method. Three temperature PCR is
the typical setting for many PCR amplifications:
♦
Template denaturation
♦
Primer annealing
♦
Primer extension
To define pre-PCR holds
:
Step Action
1 On the Create screen, in the Hld field, type in the number of pre-PCR holds for your
method.
Note You can enter 0 in this field to delete the pre-PCR hold segment from your
method.
2 Create the first temperature parameter:
a. Press
Enter
.
b. Type in a temperature value between 4.0 °C and 99.9 °C.
3 Create the first hold-time parameter:
a. Press Enter .
b. Type in a hold-time value between 00:00 and 98:59 (minutes:seconds).
4 Enter information for the next pre-PCR hold:
a. Press
Enter
.
b. Repeat step 2 and step 3 until you have hold-time and temperature values for
each of the pre-PCR hold parameters you defined in step 1.
To define the PCR parameters:
Step Action
1 On the
Create
screen, in the
Tmp
field, type in the number of temperature control
parameters (2–6) you want for the PCR segment of your method.
Note Specifying only the minimum number of PCR cycles required for analysis
will minimize the chance that unwanted targets will amplify competitively.
2 Enter the number of cycles you want the method to run:
a. Press
Enter
to select the
Cycles
field.
b. In the Cycles field, type in the number of cycles (from 2–99).
Note Twenty-five cycles is the default setting.
3 Create the first temperature parameter:
a. Press
Enter
.
b. Type in a temperature value between 4.0 °C and 99.9 °C.