8.

Preparation of working amplicon dilutions

8.1. Assessment of amplicon concentrations In:

<Amplicon Conc’n>
Tabulate the measured concentrations in the ______SHEET. from 7.3.
<Diluted amplcon>
from 8.4.
Less than 3.75 to 33.3 33.34 to 99.9 Greater than
Concentration
3.75 ng/ul ng/ul ng/ul 100 ng/ul Out:
<Amplicon DF
Target dilution assessment> to 8.3
Assessment Reject Dilute 1:5 Dilute 1:10
range <Working dilutions>
to 9.1.
Export the _____ SHEET. Lists down samples needing 5-fold or 10-fold dilutions

8.2. Prepare diluent volumes Out:

5DF/10DF diluents to
For the 5-fold dilutions, aliquot 16 ul sddH2O to the wells of 8-tube strips 8.3

For the 10-fold dilutions, aliquot 18 ul sddH2O to the wells of 8-tube strips

8.3. Amplicon dilution In: Purified amplicons

from 6.3.
For amplicons ranging between 33.33 to 100 ng/ul, transfer 4 ul amplicon to a 5-fold dilution well. <Amplicon DF
assessment> from 8.1
Pipet mix 10 times and spin down.
5DF/10DF diluents
from 8.2
For amplicons more than 100 ng/ul, transfer 2 ul amplicon to a 10-fold dilution well. Pipet mix 10
Out: Dil amplicon to
times and spin down. 8.4. 10.2.

8.4. Measurement and Reassessment In:

Dye aliquots from 7.1.
Transfer 1 μl of a dilution to an aliquot of ONE dsDNA Dye. Diluted amplicons to
8.4.
Vortex mix, spin down and let stand at RT for 2 mins.
Out:
Load a sample tube on to the device, press down lid to measure and record amplicon concentration. <Diluted amplicon> to
8.1
Using the criteria in 8.1., continue performing 8.2. to 8.4. until concentration falls in the acceptable
range (3.83 to 33.33 ng/ul)

In the ______SHEET, tabulate the eligible samples, the dilution factor performed, and the measured
concentration of the usable dilution

Store at 4°C if clean-up will be performed immediately. #EEF1F1