The BioSprint 15 (BS 15) workstation is an open extraction platform for processing of up to 15 samples per run using 5-tube plastic strips (Qiagen). The system is identical to the KingFisher™ mL which is produced by Thermo Fisher Scientific, Inc. The extraction was performed as described before for the KF Duo using the respective protocol described in Table 1 . For details on pre-filling of 5-tube strips see Additional file 1 : Table S1. The total running time to complete the BS 15 extraction was 7.5 min.
Kingfisher ml
Manufactured by Thermo Fisher Scientific
Sourced in Netherlands
The KingFisher™ mL is a magnetic particle processor designed for automated sample preparation. It can perform nucleic acid and protein purification, cell separation, and other sample preparation workflows. The instrument uses magnetic particles to capture, wash, and elute target analytes from various sample types.
Automatically generated - may contain errors
4 protocols using kingfisher ml
1
Open-Source Automated DNA Extraction
2
Virus Particle Concentration and Nucleic Acid Extraction
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Samples were processed within one week after sampling using the procedure as previously described (Medema et al., 2020a (link)). In short, centrifugation was used as pre-treatment to remove larger particles. Virus particles were concentrated from 50 ml supernatant by ultrafiltration through Centricon® Plus-70 centrifugal ultrafilters with a cut-off of 30 kDa (Millipore, Amsterdam, Netherlands). Mouse Hepatitis Virus (MHV)-A59 (Department Medical Microbiology, Leiden University Medical Center, Leiden, Netherlands) was spiked to each concentrate as quality control. Nucleic acid was extracted from the concentrate with the Biomerieux Nuclisens kit (Biomerieux, Amersfoort, Netherlands) in combination with the semi-automated KingFisher ml (Thermo Scientific, Bleiswijk, Netherlands) as previously described. Extracted nucleic acid was eluted in a volume of 100 μl.
3
Isolation and Characterization of Dental Pulp Stem Cells
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DPSCs were isolated from the incisor dental pulp of 3 months old Wistar rats (n = 4 pulp samples in each extraction). Cell isolation and characterisation were performed, as previously described (Alksne et al., 2019) . Briefly, dental pulp samples were washed several times with IMDM supplemented with 100 µg/mL Primocin™ and mechanically minced into < 1 mm 3 fragments that were transferred to the digestive solution (0.5 % collagenase type 1, 0.3 % hyaluronidase, 0.25 % trypsin and 0.02 % EDTA) and were shaken for 30-45 min at 37 °C. Later on, IMDM supplemented with 10 % PBS and antibiotic (100 U/ mL penicillin, 100 mg/mL streptomycin), referred to as GM, was added and centrifuged twice at 300 ×g for 10 min (CL10 centrifuge Thermo Scientific). The supernatant was removed, and the cells were seeded in GM. When cells reached 70-80 % confluence, CD44positive cells were extracted using magnetic beads www.ecmjournal.org coated with antibodies against the CD44 antigen. This separation procedure was performed according to BioLab's magnetic beads recommendations using the KingFisher™ mL (Thermo Scientific) purification system. Cells used in the experiments were up to 12 passages. Cells were cultivated in IMDM supplemented with 10 % PBS and antibiotics (100 U/mL penicillin, 100 mg/mL streptomycin) at 37 °C, 5 % CO 2 environment.
4
Viral RNA Extraction and Purification
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Samples were transported to the laboratory and processed as previously described (Medema et al., 2020 (link), Medema et al., 2020 (link), Medema et al., 2020 ). In short, centrifugation was used to pellet larger particles, viral particles were concentrated from 50 ml supernatant by ultrafiltration through Centricon® Plus-70 centrifugal ultrafilters with a cut-off of 30 or 100 kDa, dependent on supplies (Millipore, Amsterdam, The Netherlands). RNA extraction was also performed on ultrafiltrated PCR grade and RNAse free distilled water (Applied Biosystems, Fisher Scientific, Landsmeer, The Netherlands) water to use as negative controls. Approximately 2 × 104 genomic RNA copies from the murine coronavirus Mouse Hepatitis Virus (MHV)-A59 (obtained from “Leids Universitair Medisch Centrum”) was spiked to each concentrate and co-isolated during the extraction procedure to monitor the possible presence of RT-PCR inhibitors and measure the recovery efficiency of the extraction procedure. Nucleic acid was extracted from the concentrate using the magnetic extraction reagents of the Biomerieux Nuclisens kit (Biomerieux, Amersfoort, the Netherlands) in combination with the semi-automated KingFisher mL (Thermo Scientific, Bleiswijk, The Netherlands) as previously described. Extracted nucleic acid was eluted in a volume of 100 μl.
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