Agilent Technologies SureCycler 8800 - Page 40

4PCR Programs
To create a PCR program using the PCR Wizard
40 Agilent SureCycler 8800 Setup and User’s Guide
2 Prim
er Information
• Enter primer information by one of two methods below. Press Ne
x
t
when finished.
•Type in your primer sequences or browse to your sequence from a
connected USB
drive.
•In the absence of sequence information, enter the melting
te
mperatures (Tm) of your primers.
The primer information is used to determine the annealing temperature
for t
he PCR program. The annealing temperature is set to 5°C below
the average Tm of the two primers. If you entered the base sequences
of the primers (rather than the Tm’s) the software calculates the Tm
for each primer based on the formula:
Tm
= 69.3 + (0.41 × GC%) – (650/N)
where GC% is the percentage of GC content in the primer sequence
and N is t
he number of nucleotides in the primer sequence
3 Product Length
• Enter the expected product length and select the units as bp or
kb.
Press Ne
xt when finished.
The Wizard uses the product length to set the elongat
ion time of the
PCR program.
4 DNA Source Information
• Specify the source of template DNA from the list and then press
Ne
x
t. The source options are:
•cDNA
•Genomic DNA
•Vector DNA
T
h
e DNA source information is used to determine the duration of the
hot start or initial
denaturation step and the elongation step.