PyroMark Q24
1. Binding of biotinylated PCR product to streptavidin coated beads – in PCR plate
In a 24-well PCR plate we want a total volume of 80 µl of beads, binding buffer, water and PCR product in each well.
10-20 µl of PCR product in each well → need 70-60 µl of beads, binding buffer and water. For the Q24 system we use 2 µl of beads and
40 µl
of binding buffer, which means that we need to adjust the volume with 28 µl of water (that is if we use 10ul of PCR product).
“Mix” for 1 well
2 µl of beads
40 µl of binding buffer
x 30
= 42 µl
→Fill up with 28 µl of water (Milli-Q)
“Mix” for 30 wells
60 µl of beads
1200 µl of binding buffer
= 1260 µl
840 µl of water (Milli-Q)
Add 70 µl of “mix” in each well in the PCR plate and then add 10 µl of the PCR product. Put lids on the wells and shake the samples 10min, 1400rpm, 25ºC.
Prepare sequence primer – in PSQ plate
Add 25 µl of 0.3 µM primer (dilute primer in annealing buffer) in each well.
2. Sample Prep – Vacuum Prep Workstation
A.
Flush with water
B.
Capture beads with Vacuum Prep Tool
C.
Flush with EtOH, 5s
D.
Denaturation solution NaOH, 5s
E.
Washing buffer 5 s
F.
Turn off vacuum
G.
Release template in PSQ plate
H.
Wash VPT in water
→ Single stranded PCR product and sequence primer in PSQ plate
I.
Annealing 80 ºC, 2min
3. Enzyme mix, substrate mix, nucleotides
Volume info from the software
Use cartridge with RDTs (reagent dispensing tip) – black
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E
T
A
S
C
G
Ready for run!
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