Techne TC-5000 - Manual (Page 32)

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Parameter 2 Step PCR 3 Step PCR RT PCR
Heated Lid 105°C 105°C 105°C
Heated lid before program On On On
Pause before program Off Off Off
Initial denaturation 05m00s 94.0°C 05m00s 94.0°C 05m00s 94.0°C
Hot start Disabled Disabled Disabled
Final extension 05m00s 72°C 05m00s 72°C 07m00s 72°C
Final hold 10°C 10°C 10°C
The thermal cycling conditions are different for each of these three templates:
2 Step PCR 3 Step PCR RT PCR
Cycles 1
Segment 40m00s 45.0°C
Segment 05m00s 94.0°C
Cycles 30 30 40
Segment 00m30s 92.0°C 00m30s 92.0°C 01m00s 94.0°C
Segment 01m00s 68.0°C 00m30s 55.0°C 00m50s 55.0°C
Segment 00m30s 72.0°C 01m00s 72.0°C
The RT PCR template is for those experiments where the starting material is RNA rather than DNA.
RNA requires reverse transcription by a reverse transcriptase enzyme into complementary DNA (cDNA)
prior to the amplification by the DNA polymerase in PCR. The extra 45°C step is for the reverse
transcription and the 94°C step is to denature the reverse transcriptase before the PCR.
WRITING AND EDITING PROGRAMS
There are three options available for creating programs on the TC-5000:
1. Copy and edit one of the pre-written templates.
2. Edit a pre-written template and save it using a different name.
3. Create a new program with user-specific parameters.
There are 5 pre-written templates. These templates cannot be altered; however, they can be copied,
given a new name and then edited to save time when writing a new program.
The Ice Bucket and Ligation 15 Degrees programs are simple temperature holds, with the former
having an infinite hold at 4°C and the latter at 15°C. In both of these programs the heated lid is
switched off and the initial denaturation, hot start and final extension functions disabled.
The 2 Step and 3 Step PCR templates along with the RT PCR template have the following default
parameters: