Agilent Technologies SureCycler 8800 Setup And User Manual page 40

4 PCR Programs
To create a PCR program using the PCR Wizard
2 Primer Information
3 Product Length
4 DNA Source Information
40
• Enter primer information by one of two methods below. Press Next
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
temperatures (Tm) of your primers.
The primer information is used to determine the annealing temperature
for the 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 the number of nucleotides in the primer sequence
• Enter the expected product length and select the units as bp or kb.
Press Next when finished.
The Wizard uses the product length to set the elongation time of the
PCR program.
• Specify the source of template DNA from the list and then press
Next. The source options are:
•cDNA
•Genomic DNA
•Vector DNA
The DNA source information is used to determine the duration of the
hot start or initial denaturation step and the elongation step.
Agilent SureCycler 8800 Setup and User's Guide