Techne TC-512 - Manual (Page 44)

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Initial denaturation: Many experimental
protocols require a high temperature
incubation to remove any secondary structure
of the nucleic acid template.
Selecting "Enable" allows a temperature and
time to be added. The cursors are used to
move between the time and the temperature.
On the left of the numbers there are 3
buttons for setting the time: h for hours, m
for minutes and s for seconds. The correct
button should be pressed after the
temperature has been typed.
➔ ➔
Edit initial denaturation setting
Initial denaturation 05m00s 94°C
•
2
5
OK
Cancel
Enable
8
0
1
4
7
s
m
h
BS
3
6
9
➔ ➔
Edit initial denaturation setting
Initial denaturation xxxxxx xxxxx°C
•
2
5
OK
Cancel
Enable
8
0
1
4
7
s
m
h
BS
3
6
9
Hot start: Many standard enzymes used in molecular biology techniques can hybridise to and extend
non-specific products, resulting in multiple bands on an agarose gel, a greatly reduced yield or no
amplification of the specific product. The 'Hot Start' programming step is used to pause the instrument
at a specific temperature, typically greater than 70ºC, after the initial template denaturation. The
reason is to allow the manual addition of unmodified Taq DNA polymerase which may loose activity if
added during the initial 5 minute denaturation. Heat-activated Taq or Hot Start enzymes do not require
this step.
Final Extension: Many PCR experiments require a final step at the end of the cycling phase to allow
the polymerase to complete the synthesis of the DNA strands. By selecting "Enable" it is possible to
put a time and temperature for the step. A typical example is 72°C for 7 minutes.
Final Hold: Frequently the samples are required for further analysis such as agarose gel electrophoresis
and the samples are better stored at a temperature lower than ambient. Selecting "Enable" allows the
samples to be held infinitely between 4 and 99°C. This step requires the user to stop the program
manually on the instrument. Samples are normally held between 4 and 10°C.
Pause before program: This allows the insertion of a pause in the experiment before the experiment
starts. After the pause there must be user intervention before the program can move on to the next
step of the program.
TC512 Techne Manual 19/12/08 3:30 PM Page 44