User manual

Next-Generation Sequencing Library Analysis
70 | Droplet Digital
™
PCR Applications Guide
3. Add 20 µl of TE buffer for each well used; add additional TE buffer by multiplying by the
number of combined replicate wells if applicable.
4. In a fume hood, add 70 µl of chloroform for each well and cap the tube. Add additional
chloroform by multiplying by the number of combined replicate wells if applicable.
5. Vortex at maximum speed for 1 min.
6. In a centrifuge, spin down at 15,500 x g for 10 min.
7. Remove the upper aqueous phase by pipetting, avoiding the chloroform phase,
and transfer it to a clean 1.5 ml tube (this is the recovered DNA).
8. Dispose of the chloroform phase appropriately.
9. If desired, estimate size using Bio-Rad’s Experion
™
DNA 1K analysis kit chip and/or
requantify by ddPCR.
In Figure 7.8, the contents in the Eppendorf tubes following the droplet breaking protocol
demonstrate the layers formed by chloroform and the broken aqueous layer. The upper
aqueous phase can readily be pipetted into a clean tube for downstream analysis.
Fig. 7.8. Aqueous phase recovery following droplet amplicon recovery protocol.
Recovered DNA can be analyzed by gel electrophoresis (Figure 7.9), sequencing,
and ddPCR (Figure 7.10).
Chloroform Aqueous