Materials

Programmable thermocycler

Not all thermocyclers are capable of performing the rather complex cycling programs required for TAIL-PC R. DNA Engines, from MJ Research, and Perkin-Elmer model 9600 machines will work. The following programs have been optimized for the MJ Research machine. Some modifications may be necessary when using other machines (for details, see Liu and Whittier 1995).

TAIL Program 1

Step 1 =

4°C for 2 minutes

Step 2 =

93°C for 1 minute

Step 3 =

95°C for 1 minute

Step 4

94°C for 30 seconds

Step 5 =

62°C for 1 minute

Step 6 =

72°C for 2 minutes and 30 seconds

Step 7

Go to Step 4 for four more cycles

Step 8

94°C for 30 seconds

Step 9

25°C for 3 minutes

Step 10 =

Ramp at 0.2°C per second to 72°C

Step 11 =

72°C for 2 minutes and 30 seconds

Step 12 =

94°C for 10 seconds

Step 13 =

68°C for 1 minute

Step 14 =

72°C for 2 minutes and 30 seconds

Step 15 =

94°C for 10 seconds

Step 16 =

68°C for 1 minute

Step 17 =

72°C for 2 minutes and 30 seconds

Step 18 =

94°C for 10 seconds

Step 19 =

44°C for 1 minute

Step 20 =

72°C for 2 minutes and 30 seconds

Step 21 =

Go to Step 12 for 14 more cycles

Step 22 =

72°C for 5 minutes

Step 23 =

4°C hold

Step 24 =

End

The slow ramping in Step 10 is slightly longer than that specified in Liu and Whittier (1995). This was determined by the programming style of the MJ machine. Because of this, a 2-minute hold at 4°C has been included at the beginning of the program.

TAIL Program 2

Step 1

: 4°C for 2 minutes

Step 2 =

= 94°C for 10 seconds

Step 3 =

= 64°C for 1 minute

Step 4

= 72°C for 2 minutes and 30 seconds

Step 5 =

: 94°C lor 10 seconds

Step 6 =

- 64°C for 1 minute

Step 7

= 72°C for 2 minutes and 30 seconds

Step 8 =

= 94°C for 10 seconds

Step 9

= 44°C for 1 minute

Step 10 =

= 72°C for 2 minutes and 30 seconds

Step 11 =

= Go to Step 2 for 11 more cycles

Step 12 =

= 72°C for 5 minutes

Step 13 =

= 4°C hold

Step 14 =

= End

TAIL Program

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