User Manual

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: