Improvement

Effect

Volume of sample

4 ml

Volume of IC DNA

4 ml (0.1 ml concentrated IC discarded at first,

0.2 ml IC in 3.8 ml sterile H2O)

SYBR Green I

Diluted 1:20 in Dimethyl sulfoxide (DMSO).

Volume equals to dead space in Pump B (20 μL)

Incubation of sample

13 min at 60˚C

Incubation of IC

Less than one minute at ambient temperature

Flow rate and time

3.5 μL・s−1 of 180 sec and 10 sec

Measurement wavelength interval

520 - 530 nm

Fluorescence count

Average of readings every one sec for 180 and 10 sec

Background reading (auto-fluorescence)

To be subtracted from sample reading. Fluorescence

reading of sample without dye, for correction of any

auto-fluorescence of the sample water. Background

reading is not subtracted from internal control readings.

Cleaning

Sterile H2O and air between sample and IC

measurement. Acid, alkaline, sterile water and air

at the end of a cycle. No air through the flow cell.

Time consumption

75 min for one full cycle, including washing/cleaning