User's Manual

14
Primer testing
For every new target and prior to generating droplets, both the dPCR enrichment primers and the qPCR QC
primers must be optimized preferably by qPCR using your sample DNA and Samplix dPCR reagents ● and
qPCR dye ●. For this assay, include a negative control with no template and at least three different
concentrations of your template (or a biological replicate) in the range of the amount of input DNA suggested
by the online sample calculation tool at samplix.com (see Fig. 2.1).
Use the Samplix primer test PCR kit (Cat. No. RE10200) that includes Samplix dPCR mix (2X) ● and Samplix
qPCR dye ● to verify the primers and reaction efficiency.
The primer test reagents are optimized for the following reaction mix using Samplix Primer test PCR kit (Cat.
No. RE10200):
dPCR reaction mix
1 reaction
H
2
O (molecular grade)
7,2 μl
dPCR mix (2x) ●
10 μl
Primer Forward 10 µM
0,4 μl
Primer Reverse 10 µM 0,4 μl
qPCR dye (20x)
●
1 μl
Total mix
19 μl
Template DNA
1 μl
Total volume
20 µl
And the following qPCR program:
Temperature
Duration
Cycles
30°C
5 sec
1x
94°C
2 min
94°C
3 sec
40x
Annealing temp.
30 sec
Data acquisition*
-
Melting curve 70-
90°C
1X
*Follow manufacturer’s protocol for data acquisition program.
Note: The annealing temperature needs to be adjusted to the specific primer sets tested
Note: qPCR QC primer can be tested either in the Samplix dPCR reagents or in your preferred qPCR reagents