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Magna pure 96 external lysis buffer

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The MagNA Pure 96 External Lysis Buffer is a solution designed for use with the MagNA Pure 96 System. It is used to lyse samples and prepare them for nucleic acid extraction and purification on the MagNA Pure 96 platform.

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Lab products found in correlation

Isothermal amplification buffer, by New England Biolabs (2 mentions) Bst 2.0 warmstart dna polymerase, by New England Biolabs (2 mentions) Warmstart rtx reverse transcriptase, by New England Biolabs (2 mentions) Magna pure 96 extraction system, by Roche (2 mentions) Mgso4, by New England Biolabs (2 mentions) 7500 real time pcr system instrument, by Thermo Fisher Scientific (1 mentions) Standard m ncov real time detection kit, by SD Biosensor (1 mentions) Abi prism 7000 sequence detection system, by Thermo Fisher Scientific (1 mentions) Dynamag 96 magnet, by Thermo Fisher Scientific (1 mentions) Taqman fast virus 1 step master mix, by Thermo Fisher Scientific (1 mentions) Agencourt ampure xp magnetic beads, by Beckman Coulter (1 mentions) Dulbecco s phosphate buffered saline dpbs, by Corning (1 mentions) Magna pure 96, by Roche (1 mentions) Magna pure 96 instrument, by Roche (1 mentions) Tpck treated trypsin, by Merck Group (1 mentions) Lysis matrix d, by MP Biomedicals (1 mentions) Rnalater, by Thermo Fisher Scientific (1 mentions) Magna pure lc instrument, by Roche (1 mentions) Magna pure 96 platform, by Roche (1 mentions) Magna pure 96 system, by Roche (1 mentions)

14 protocols using magna pure 96 external lysis buffer

1

SARS-CoV-2 qRT-PCR Detection Protocol

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Total RNA was extracted using a MagNA Pure 96 External lysis buffer (Roche, Switzerland), and qRT-PCR reactions were performed using STANDARD M nCoV Real-Time Detection kit (M-NCOV-01, SD Biosensor, KOREA), which targeted regions of envelope (E) and RNA-dependent RNA polymerase (RdRp) [14 (link)] according to the manufacturer’s protocol. Briefly, PCR were run at an Applied Biosystems 7500 Real-Time PCR instrument system in a volume of 31 μL containing a 10 μL sample, a 14 μL 2019-nCoV reaction solution, 6 μL RTase mix, 0.5 μL ROX, and 0.5 μL internal control. The PCR conditions were as following: 15 min at 50 °C for reverse transcription, 3 min at 95 °C for initial denaturation, 5 cycles of 5 s at 95 °C and 40 s at 60 °C for pre-amplification, and 40 cycles of 5 s at 95 °C and 40 s at 60 °C for amplification. Viral RNA was shown as a cycle threshold (Ct) value that is inversely proportional to the original relative expression level of the target gene.
Jeong S.K., Heo Y.K., Jeong J.H., Ham S.J., Yum J.S., Ahn B.C., Song C.S, & Chun E.Y. (2021). COVID-19 Subunit Vaccine with a Combination of TLR1/2 and TLR3 Agonists Induces Robust and Protective Immunity. Vaccines, 9(9), 957.
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2

RNA Extraction and qRT-PCR for NDV Detection

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RNA was extracted from the urine, fecal and throat samples by adding 60 μl sample to 90 μl Magnapure 96 external lysis buffer (6374913001, Roche Diagnostics, The Netherlands) as described before (Richard et al., 2020 (link)). Subsequently, the lysed sample was added to 60 μl Agencourt AMPure XP magnetic beads (A63880, Beckman Coulter, The Netherlands) and incubated 15 min at room temperature. Magnetic beads were washed three times with 70 % ethanol using the DynaMag-96 magnet (12027, Invitrogen, The Netherlands) and subsequently air-dried. RNA was eluted by 6 min of incubation in bidest H2O. NDV-specific quantitative reverse transcription-PCR was performed using 5 μl RNA in an ABI PRISM 7000 Sequence Detection System using TaqMan Fast Virus 1-Step Master Mix (both from Thermo Fischer) in a total volume of 30 μl. The NDV-specific primers used were described by Wise et al. (2004) (link). The reverse transcriptase step was 5 min at 50 °C, followed by 95 °C for 20 s. Cycling consisted of 40 cycles of 3 s denaturation at 95 °C, 5 s annealing at 54 °C and 31 s extension at 60 °C.
de Graaf J.F., Huberts M., Groeneveld D., van Nieuwkoop S., van Eijck C.H., Fouchier R.A, & van den Hoogen B.G. (2022). Comparison between intratumoral and intravenously administered oncolytic virus therapy with Newcastle disease virus in a xenograft murine model for pancreatic adenocarcinoma. Heliyon, 8(7), e09915.
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3

Cobas 4800 SARS-CoV-2 Detection Protocol

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The Cobas 4800 is a fully automated instrument that allows extraction of nucleic acids from samples, followed by real-time polymerase chain reaction (PCR) amplification and detection. Prior to loading 96 samples onto the Cobas® 4800 system for extraction, MagNA Pure 96 external lysis buffer (Roche Diagnostics, Mississauga, Ontario, Canada) is added at a ratio of 1:1 and 400 µl is tested for each sample. Due to the lack of user-defined workflow for SARS-CoV-2 on the Cobas® 4800 system, the CT/NG user-defined workflow (under “test type” in the system's software) was selected instead following the manufacturer's recommendation. Once each extraction was completed, the original 96-well plate is discarded, but the corresponding “deep-well” plate containing the purified nucleic acids is conserved for the remaining steps in the SARS-CoV-2 amplification and detection process.
LightMix® SarbecoV E-gene plus EAV control assay is performed (Roche Diagnostics, Mississauga, Ontario, Canada), as described in Tib Molbiol document MDx 40-0776-96-V200422. Cobas 4800 software version 2.2.0.1509 was used for extraction and Cobas user-defined workflow (UDF) software version 2.0.1 was used for PCR.
Baksh S., Volodko N., Soucie M., Geier S.B., Diep A., Rozak K., Chan T.Y., Mustapha J., Lai R., Estey M., Verity B, & Lee M.C. (2021). Extractionless nucleic acid detection: a high capacity solution to COVID-19 testing. Diagnostic Microbiology and Infectious Disease, 101(2), 115458.
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4

Vaginal Microbiome Characterization Protocol

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Vaginal swabs, collected by clinical staff, were obtained from healthy women during routine gynecological examinations using a sterile calcium alginate swab (Fisherbrand, Pittsburgh, PA) passed over the mid-vaginal wall avoiding external sites via the placement of a standard speculum. The swabs were immediately placed into 2ml of sterile, Ca++/Mg++-free Dulbecco’s Phosphate Buffered Saline (DPBS; Cellgro, Herndon, VA), and transported at 4°C to the lab. The subsequent procedures were all completed in a sterile class II biosafety cabinet. Each swab was thoroughly vortexed to release the collected bacteria and human cells and then aliquoted for DNA and RNA separately into MagNA Pure 96 External lysis buffer (Roche, Basel, Switzerland) for molecular evaluations or mixed with sterile glycerol (10% w/v) as a cryo-protectant for viable VMB community aliquots. All aliquots were stored at −80°C until utilized. DNA or RNA was extracted on an automated MagNA Pure 96 (Roche). VMBs were characterized by customized qPCR array as described previously [17 (link)]. Any VMB samples molecularly positive for STI, yeast or Y chromosome were excluded from the study.
Medina-Colorado A.A., Vincent K.L., Miller A.L., Maxwell C.A., Dawson L.N., Olive T., Kozlova E.V., Baum M.M, & Pyles R.B. (2017). Vaginal ecosystem modeling of growth patterns of anaerobic bacteria in microaerophilic conditions. Anaerobe, 45, 10-18.
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5

USUV Detection in Blackbird Samples

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For USUV, molecular detection was performed as described previously by Oude Munnink et al. 2020 [33 (link)]. Brain tissue samples (N = 200) collected from blackbirds during post-mortem investigation were homogenized in 300uL tissue lysis buffer (MagNA Pure DNA Tissue Lysis Buffer) using the Fastprep bead beater (4.0 m/s for 10 s), RNA extraction and USUV RT-PCRs were performed with an additional dilution step, where 60uL homogenized sample was added to 540uL VTM (or DMEM) and 600uL External lysis buffer. Phocine distemper virus (PDV) was used as an internal control [34 (link),35 (link)]. Throat and cloaca swabs of live blackbirds were stored in Virus Transport Medium (VTM). 600 uL of MagNA Pure 96 External Lysis Buffer (Manufactured by Roche: https://lifescience.roche.com/en_nl/products/magna-pure-96-external-lysis-buffer.html) was added to 600 uL of sample and RNA extraction was performed on a MagNA Pure 96 Instrument (MagNA Pure 96 Instrument (roche.com)). USUV RT-PCRs [34 (link),35 (link)] were performed, using Phocine distemper virus (PDV) as an internal control. For USUV investigation, mosquito pools were tested according to the protocol reported by Blom et al., 2023 [36 (link)].
Agliani G., Giglia G., de Bruin E., van Mastrigt T., Blom R., Sikkema R.S., Kik M., Koopmans M.P., Gröne A, & Van den Brand J.M. (2023). The pathology of co-infection with Usutu virus and Plasmodium spp. in naturally infected Eurasian blackbirds (Turdus merula). One Health, 16, 100534.
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6

Synchronized Viral Entry Kinetics

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Synchronized infection experiments were performed to determine entry efficiency of the virus variants. Infection of cells was performed on ice and cells were immediately transferred to 4°C for 1 hour after virus dilutions were added to ensure synchronized virus uptake and start of replication. After virus adsorption, cells were washed 5 times with PBS to remove excess virus particles. Cells were lysed either immediately or incubated with infection medium until 4 or 6 hours postinfection. At the indicated time points, medium was removed and cells were lysed with MagNA Pure 96 external lysis buffer (Roche, Penzberg, Germany). Isolation of RNA from cell lysates and quantitative RT-PCR on subgenomic nucleocapsid RNA was performed as described elsewhere [69 (link),70 (link)]. Entry inhibitors were dissolved in DMSO in the indicated concentrations, added 1 hour prior to virus infection, and were supplied for the entire duration of the experiment. Pretreatment of virions with TPCK-treated trypsin (Sigma Aldrich) or saliva (1:10 diluted in serum-free medium) was performed for 1 hour at 37°C prior to virus infection.
Niemeyer D., Stenzel S., Veith T., Schroeder S., Friedmann K., Weege F., Trimpert J., Heinze J., Richter A., Jansen J., Emanuel J., Kazmierski J., Pott F., Jeworowski L.M., Olmer R., Jaboreck M.C., Tenner B., Papies J., Walper F., Schmidt M.L., Heinemann N., Möncke-Buchner E., Baumgardt M., Hoffmann K., Widera M., Thao T.T., Balázs A., Schulze J., Mache C., Jones T.C., Morkel M., Ciesek S., Hanitsch L.G., Mall M.A., Hocke A.C., Thiel V., Osterrieder K., Wolff T., Martin U., Corman V.M., Müller M.A., Goffinet C, & Drosten C. (2022). SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression. PLOS Biology, 20(11), e3001871.
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7

RT-LAMP Assay for COVID-19 Detection

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TNA extraction was performed using the MagNA Pure 96 extraction system (Roche, Switzerland) according to the manufacturer’s instructions, as we described previously (7 (link)). Briefly, 200 μL of each sample was mixed with MagNA Pure 96 external lysis buffer (Roche). After extraction, TNA was recovered in 50 μL of elution buffer and then kept at −80°C until use.
Before the preparation of a master mix for the reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay, 10× LAMP primer mix was prepared (see Table S3 in the supplemental material). The RT-LAMP reagent mixture (10 μL) contained 0.4 μL of nuclease-free water, 1 μL of 10× isothermal amplification buffer (NEB, USA), 0.6 μL of 100 mM MgSO4 (NEB), 1.4 μL of 10 mM dNTP solution mix, 1 μL of 10× LAMP primer mix, 0.4 μL of Bst 2.0 WarmStart DNA polymerase (8,000 U/mL) (NEB), 0.2 μL of WarmStart RTx reverse transcriptase (15,000 U/mL) (NEB), and 5 μL of TNA as the template. RT-LAMP reactions were performed at 60°C for 40 min for the COVID-19 nsp8 assay, at 62°C for 30 min for the COVID-19 N assay, and at 62°C for 40 min for the human RNase P assay.
Yip C.C., Sridhar S., Chan W.M., Ip J.D., Chu A.W., Leung K.H., Cheng V.C., Yuen K.Y, & To K.K. (2022). Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis. Microbiology Spectrum, 10(6), e01962-22.
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8

RT-LAMP Assay for COVID-19 Detection

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TNA extraction was performed using the MagNA Pure 96 extraction system (Roche, Switzerland) according to the manufacturer’s instructions, as we described previously (7 (link)). Briefly, 200 μL of each sample was mixed with MagNA Pure 96 external lysis buffer (Roche). After extraction, TNA was recovered in 50 μL of elution buffer and then kept at −80°C until use.
Before the preparation of a master mix for the reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay, 10× LAMP primer mix was prepared (see Table S3 in the supplemental material). The RT-LAMP reagent mixture (10 μL) contained 0.4 μL of nuclease-free water, 1 μL of 10× isothermal amplification buffer (NEB, USA), 0.6 μL of 100 mM MgSO4 (NEB), 1.4 μL of 10 mM dNTP solution mix, 1 μL of 10× LAMP primer mix, 0.4 μL of Bst 2.0 WarmStart DNA polymerase (8,000 U/mL) (NEB), 0.2 μL of WarmStart RTx reverse transcriptase (15,000 U/mL) (NEB), and 5 μL of TNA as the template. RT-LAMP reactions were performed at 60°C for 40 min for the COVID-19 nsp8 assay, at 62°C for 30 min for the COVID-19 N assay, and at 62°C for 40 min for the human RNase P assay.
Yip C.C., Sridhar S., Chan W.M., Ip J.D., Chu A.W., Leung K.H., Cheng V.C., Yuen K.Y, & To K.K. (2022). Development and Validation of a Novel COVID-19 nsp8 One-Tube RT-LAMP-CRISPR Assay for SARS-CoV-2 Diagnosis. Microbiology Spectrum, 10(6), e01962-22.
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9

Stratified Vaginal Epithelial Multilayer Model

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Immortalized human VEC (V19I and BVEC02I) were cultured as described [20 (link)] and used to produce stratified squamous vaginal multilayers [17 (link), 18 (link)]. Briefly, VEC from one of 2 donors were plated in 24 (106 cells/transwell) or 96 (105 cells/transwell) well transwell format and cultured at 37°C with 5% CO2. After monolayer formation, each culture was subjected to air-interfacing by removal of the apical growth medium. The multilayers were refed basally every other day with antibiotic- and serum-free medium as described [17 (link)] leading to stratification and differentiation of multilayers.
At maturation (7–9 days) VMB were diluted to 103-104 total genomes and applied to the apical surface of the multilayers. VMB were selected from our cryo-repository based on community profiles established from initial swab evaluations. In supplementation studies we used 1,000 cfu of our previous L. jensenii clinical isolate [18 (link)]. Parallel triplicate cultures were created and immediately placed in prepared 37°C, humidified incubators containing AA supplemented with either 5% or 10% CO2. The cultures were maintained for 48h before harvesting in MagNA Pure 96 External lysis buffer (Roche). Each study was repeated at least once.
Medina-Colorado A.A., Vincent K.L., Miller A.L., Maxwell C.A., Dawson L.N., Olive T., Kozlova E.V., Baum M.M, & Pyles R.B. (2017). Vaginal ecosystem modeling of growth patterns of anaerobic bacteria in microaerophilic conditions. Anaerobe, 45, 10-18.
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10

SEOV Detection in Diverse Samples

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Lung, kidney and liver tissues were collected in RNAlater (Applied Biosystems, Foster City, CA, USA) and stored at −80 °C. Tissue samples (+/− 25 mg per sample) were disrupted in MagNA Pure 96 External Lysis Buffer (Roche) by using Lysis matrix D (MP Biomedicals, Santa Ana, CA, USA) and Fast Prep FP120 Homogenizer (Thermo Savant, Carlsbad, CA, USA). RNA isolation was performed on blood (200 µL), urine (200 µL), saliva and rectal swabs using a QIAamp Viral RNA kit. All samples were tested by a SEOV–specific real-time RT-PCR as previously described [6 (link)]. Equine Arteritis Virus was added in the lysis buffer to all samples prior to nucleic acid extraction to exclude inhibitory factors in the sample itself as quality assurance for each experimental step [19 (link)]. A rat β-actin control was used as an amplification control by conventional RT-PCR, to confirm the extraction of nucleic acid from tissue material. Only animals with samples positive for β-actin were included.
Maas M., van Heteren M., de Vries A., Kuiken T., Hoornweg T., Veldhuis Kroeze E, & Rockx B. (2019). Seoul Virus Tropism and Pathology in Naturally Infected Feeder Rats. Viruses, 11(6), 531.
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