Magnesil paramagnetic particles pmps

DNA extraction was performed as previously described^{44 (link)}. DNA was extracted using a semi-automated Gilson PIPETMAX liquid handling robot enabled for exclusion-based sample preparation (ESP), termed EXTRACTMAX^{60 (link)}. The robot added LiDS buffer (90 mM Tris-HCL, 500 mM lithium chloride, 1% Igepal CA-630, 10 mM EDTA, 1 mM dithiothreitol) and MagneSil Paramagnetic Particles (PMPs) (Promega, Cat# MD1441) resuspended in GTC buffer (10 mM Tris-HCl, 6 M guanidinium thiocyanate, 0.1% Igepal CA-630, pH 7.5) to the extraction microplate (Gilson, Cat# 22100008). The robot then adds cells in suspension to the well-containing LiDS, GTC, and MagneSil beads. Cells are mixed in the buffer by the robot. Cells were lysed and DNA was allowed to bind to MagneSil PMPs for 5 min. The MagneSil PMPs with bound DNA were robotically transferred by exclusion liquid repellency (ELR) through one PBST (PBS containing 0.1% Tween-20) wash, one PBS wash, and eluted into 15 µL of nuclease-free water (Promega, Cat# P1197). DNA was eluted off of beads for 2 min following manual resuspension. The robot then transferred the MagneSil PMPs out of the elution well, leaving the eluted DNA in water.

Semi-automated DNA extraction was performed on a Gilson PIPETMAX liquid handling robot enabled for exclusion-based sample preparation (ESP), termed EXTRACTMAX [50 (link)]. LiDS buffer (90 mM Tris–HCL, 500 mM lithium chloride, 1% Igepal CA-630, 10 mM EDTA, 1 mM dithiothreitol) and MagneSil Paramagnetic Particles (PMPs) (Promega) resuspended in GTC buffer (10 mM Tris–HCl, 6 M guanidinium thiocyanate, 0.1% Igepal CA-630, pH 7.5) are added to the EXTRACTMAX extraction microplate (Gilson) by the robot. Cells were added to the microplate well containing LiDS, GTC, and MagneSil beads and mixed by the robot. Cells were allowed to lyse, and DNA was allowed to bind to MagneSil PMPs for 5 min. The robot then transferred the MagneSil PMPs with bound DNA by exclusion liquid repellency (ELR) through one PBST (PBS containing 0.1% Tween-20) wash, one PBS wash, and into water for elution. Beads were manually resuspended in the elution well and allowed to elute for 2 min. The MagneSil PMPs are magnetically transferred out of the elution well, leaving eluted DNA in water. LNCaP and WBC DNA for restriction enzyme and pre-amplification validation experiments were extracted using the AllPrep DNA/RNA mini kit (Qiagen) according to manufacturer’s instructions.

Blood was collected and processed as previously described [35 (link), 51 (link)]. Briefly, whole blood collected by venipuncture into EDTA tubes was separated by centrifugation with Ficoll-Paque PLUS (Fisher Scientific). The layer containing mononucleated cells was depleted of CD45+ cells by magnetic LS MACS columns (Miltenyi). CTCs were isolated using an anti-EpCAM goat polyclonal antibody (R&D Systems). RNA was isolated using oligo (dT) Dynabeads (Invitrogen), and DNA was isolated using MagneSil Paramagnetic Particles (PMPs) (Promega) as previously described [34 (link)].