Magnesil pmps

To assess the amount of fluid carryover
in the SLIDE device, an acridine orange solution was made at a concentration
of 0.5 mg/mL in stock solutions of 0%, 0.1%, and 1% Triton X-100 in
DI water. For each experiment, 2 μL of Magnesil PMPs (#MD1471,
Promega, Madison, WI) was added to each input solution of 40 μL.
Droplets of deionized water were used as the output droplet. To evaluate
the amount of carryover, an acridine orange dilution curve was created,
and a linear fit was used to calculate the percent carryover based
on the arbitrary intensity units measured using a Qubit Fluorometer
with an excitation wavelength of 430–495 nm (Life Technologies).

Samples to measure
DNA extraction were
prepared by lysing LNCaP cells in Buffer RLT (Qiagen) for 5 min at
room temperature with 2 μL of MagneSil PMPs (Promega). Lysates
were prepared at concentrations of 1000 and 10 000 cells per
50 μL device input volume. Lysates were loaded onto SLIDE and
processed as previously described. DNA was eluted from the PMPs in
nuclease free water. As a comparison, other aliquots of this sample
were purified using a conventional technique, where PMPs were captured
against the side of a 1.5 mL microcentrifuge tube, the supernatant
was removed, and the PMPs were resuspended in buffer (Promega Wizard
Kit Wash Buffer). In the comparison samples, this wash process was
repeated four times. Extracted DNA was amplified and quantified using
qPCR on a LightCycler 480 (Roche) thermal cycler. Isolated DNA was
mixed with 2× Taqman Gene Expression Master Mix (Life Technologies)
and a commercially available assay for GAPDH genomic DNA (#4331182,
Life Technologies). The thermal cycler ran 40 cycles of 60 °C
for 1 min and 95 °C for 15 s, and threshold cycles (C_T) were calculated by the LightCycler software using the
second derivative algorithm.