7.

Amplicon Assessment

7.1 QuantiFluor Dye aliquot preparation Out:

Dye aliquots to 7.2.,
Prepare forty 0.5-mL thin-walled PCR tubes and label S for standard, B for blank, and nos. 01 to 38 8.4, 15.2

In an unlit hood and away from direct light, aliquot 199 μl ONE dsDNA Dye to the S tube and 200 μl
to all the rest

7.2. Standards and samples preparation In:

Dye aliquots from 7.1.
Purified amplicons
IMPORTANT from 6.3.

Equilibrate the QuantiFluor Dye aliquots to room temperature before using

Out:
Sample for measuring
Add 1 μl ONE Lambda DNA standard to the S tube to 7.3

Transfer 1 μl of amplicon eluate or negative controls to the corresponding labeled sample tube with
ONE dsDNA Dye (from Step 6.3 Elution)
Vortex mix, spin down and let stand at RT for 2 mins

7.3. Measurement and recording In:

Sample for measuring
Set up Quantus Fluorometer from 7.2

Before loading any tube, wipe tube surfaces with line-free wipes’ Out:
<Amplicon Conc’n> to
Calibration 8.1

Load the B tube (blank solution) and read as blank

Load the S tube (standard solution) and read as standard

Sample measurement
Load a sample tube on to the device, press down lid to measure and record amplicon concentration

NOTE NEC and NTC reactions that record <1 ng/ul are acceptable

IMPORTANT
Decontamination with UV and or DNAzap between sample batches is recommended