Applied Biosystems GeneAmp PCR System 9700, User Manual

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Supplied Methods B-3
Utilizing hot start techniques helps to minimize the formation of primer-dimers or
non-specific products, thus increasing specificity and sensitivity of PCR.
You can find further information on AmpliTaq Gold DNA Polymerase in the product
insert (P/N N808-0241) or at the Applied Biosystems website.
BigDye
The BigDye
ä
method consists of cycle sequencing parameters for dideoxy (Sanger)
terminator sequencing using ABI P
RISM®
BigDye™ Terminator Cycle Sequencing
Ready Reaction Kits (available from Applied Biosystems). It consists of 25-cycle,
three-temperature cycle sequencing followed by an infinite hold at 4 °C.
The pre-coded conditions in the BigDye
ä
method are optimized for AmpliTaq
®
DNA
Polymerase, FS and the cycle sequencing reagents supplied with the BigDye
terminator kits. This process is further described in the ABI P
RISM
BigDye Terminator
Cycle Sequencing Ready Reaction Kits Protocol (P/N 4303237).
General PCR
The General PCR method is a basic one and can be easily modified with both pre-
and post-PCR holds.
96.0
0:10 50.0
0:05
1 Hld 3 Tmp 25 Cycles
F1 F3 F4 F5 F2
Start
Return
Method: BigDye™
60.0
4:00
4.0
4.0
72.0
0:30
55.0
0:15
95.0
0:15
95.0
1.00
1 Hld 3 Tmp 35 Cycles 2 Holds
Start
Return
Method: General PCR
F1 F2 F3 F4 F5
72.0
7:00