Applied Biosystems 2720 User Manual

Supplied Methods B-3
BigDye
®
Terminator
The BigDye
®
Terminator protocol is used for cycle sequencing. It consists of
3-temperature PCR for 25 cycles followed by an infinite hold at 4 °C.
The reagents used include the sequencing enzyme AmpliTaq
®
DNA Polymerase, FS,
and fluorescent labeled dye terminators. Four different fluorescent dyes, one for each
base, are incorporated into extension products. These products can then by purified.
Subsequently, electrophoresis and data analysis can be performed on them. This
process is further described in the protocol ABI P
RISM
®
BigDye
®
Terminator Cycle
Sequencing Ready Reaction Kits (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.
Time Release PCR
The Time Release PCR method is designed for use with AmpliTaq Gold DNA
Polymerase. The enzyme is activated more slowly than with the AmpliTaq Gold
method. Here the pre-PCR hold is only 1 minute, and the number of cycles is
increased to 40.
96.0
0:10
50.0
0:05
1 Hld3 Tmp 25 Cycles
F1 F3 F4 F5F2
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
4.0
72.0
0:30
55.0
0:15
95.0
0:15
95.0
1.00
1 Hld 3 Tmp 40 Cycles 2 Holds
Start
Return
Method: Time Release PCR
F1 F2 F3 F4 F5
72.0
7:00

Applied Biosystems 2720 Specifications

General

Brand	Applied Biosystems
Model	2720
Category	Laboratory Equipment
Language	English