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Eppendorf Mastercycler ABI Big Dye Sequencing Protocol

From MAIN MENU go to FILES/ EDIT/ STANDARD rewrite accordingly then EXIT/ YES (use SEL to choose)/ ENTER/ change name to BIGDYE/ ENTER
Step
Parameter
Explanation
Additional Comments



CNTRL= TUBE LID= 105 NO WAIT AUTO
Block temperature control

Heated lid temperature

Program starts immediately
Lid shuts off at end of program
As instructed in Pharmacias Cycle
Sequencing Kit, preheat block to 95C:

Version 2.1 or above use the incubate mode
Versions 2.1 or less must include a T= 95C
HOLD step prior to step 1 remember to alter
the GO TO step accordingly!
1
T=95.0 0:02:00
+0.00 +0:00
R=1.0/s +0.0/s
G=00.0
Hold 95C for 2 min.
Change ramp rate to 1C/ sec
Initial denaturation temperature ramping at
1C/ second
2
T=95.0 0:00:30
+0.00 +0:00
R=1.0/s +0.0/s
G=00.0
Hold 95C for 30 sec.
Change ramp rate to 1C/ sec
Denaturation temperature ramping at 1C/
second
3*
T=55.0 0:00:36
+0.00 +0:00
R=1.0/s +0.0/s
G=00.0
Hold 55C for 36 sec.
Change ramp rate to 1C/ sec
Annealing temperature ramping at 1C/
second
4
T=72.0 0:00:30
+0.00 +0:00
R=1.0/s +0.0/s
G=00.0
Hold 72C for 30 sec.
Elongation temperature ramping at 1C/
second
5
GO TO 2 REP 24
Repeat cycle 24 times
Step 2-4, N = 25
6
T=72.0 0:00:30
Hold 72C for 30 seconds
Final elongation temperature
7
SOUND 05
Beep 5 times
Notification of program end- add Stop Solution
8
HOLD 4 ENTER
Hold 4C
User must end program by STOP/ ENTER
END
Use SEL key to change, add, or delete a program line.
Use OPT key to view all parameters (i.e. ramp rate).
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Source:


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