Total nucleic acids were purified using the Bio-On-Magnetic-Beads (BOMB) approach63 (link). Briefly, a guanidine isothiocyanate lysis buffer was used to homogenise cells and then was combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in a volumetric ratio of 2:3:4 (beads:lysate:isopropanol). Beads were captured with a neodymium magnet and washed once with isopropanol, twice with 70% ethanol and resuspended in milliQ water.
Sera mag magnetic speedbeads
Manufactured by GE Healthcare
Sera-Mag Magnetic SpeedBeads are a type of magnetic beads used in various laboratory applications. They are designed to enable efficient separation and isolation of target molecules or cells from complex samples. The core function of Sera-Mag Magnetic SpeedBeads is to provide a versatile and reliable solution for magnetic separation and purification processes.
Automatically generated - may contain errors
4 protocols using sera mag magnetic speedbeads
1
Nucleic Acid Extraction via BOMB
2
Barcoding and Sequencing of Oligonucleotides
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Oligonucleotides with attached MiSeq adaptors and barcodes were used for the barcoding reaction (Fluidigm PN FLD-100-3771). Barcoding was performed using 1x Green GoTaq Flexi Buffer, 0.05 U/μL of GoTaq Hot Start Polymerase, 0.25x CES, 4.5mM MgCl2, 200μM of each dNTP, 25μL of the pooled template after Sera-Mag Magnetic SpeedBeads (GE Healthcare Life Sciences) clean up. Cycling conditions for barcoding were as follows 94° C/5mins; 9 cycles of 97° C/15 seconds, 60° C/30 seconds and 72° C/2mins; 72° C/2mins; 6° C/5mins. Barcoding was performed using an Eppendorf ProS 96 or 384 thermocycler. Sequencing was performed on an Illumina MiSeq using the MiSeq Reagent Kit v2 (300 cycle; PN MS-102-2002).
3
Efficient DNA Extraction from Shark Tissues
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Elephant shark tissue samples were sourced as by-product of deceased animals harvested from commercial fishing in the Otago coastal region. As such, no animal ethics permission was applicable in this circumstance. No animal experimentation or manipulation was undertaken as defined by the Animal Welfare Act (2009, New Zealand), or according to guidelines issued by the New Zealand National Animal Ethics Advisory Committee (NAEAC, Occasional Paper No 2, 2009, ISBN 978-0-478-33858-4).
DNA was purified using a modified magnetic bead approach
44 (link). Briefly, cells were first homogenised in “GITC” lysis buffer (4 M Guanidine thiocyanate, Sigma G6639; 50 mM Tris, Thermo 15568-025; 20 mM EDTA; Thermo 15575-020; 2% Sarkosyl, Sigma L9150-50G; 0.1% Antifoam, Sigma A8311-50ML), and this lysate mixture was then combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in a volumetric ratio of 2:3:4, respectively. Following capture with a neodymium magnet, beads were washed once with isopropanol, twice with 70% ethanol and resuspended in filter-sterile milliQ water.
DNA was purified using a modified magnetic bead approach
44 (link). Briefly, cells were first homogenised in “GITC” lysis buffer (4 M Guanidine thiocyanate, Sigma G6639; 50 mM Tris, Thermo 15568-025; 20 mM EDTA; Thermo 15575-020; 2% Sarkosyl, Sigma L9150-50G; 0.1% Antifoam, Sigma A8311-50ML), and this lysate mixture was then combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in a volumetric ratio of 2:3:4, respectively. Following capture with a neodymium magnet, beads were washed once with isopropanol, twice with 70% ethanol and resuspended in filter-sterile milliQ water.
4
Rapid RNA Purification using SPRI Beads
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Solid-phase reversible immobilisation (SPRI) on carboxylated paramagnetic beads (Sera-Mag Magnetic SpeedBeads, from GE Healthcare) were prepared for RNA binding as described (https://openwetware.org/wiki/SPRI_bead_mix ). RNA purification was performed in 96-well plates with initial lysis by the addition of 25 µl of 6GTD lysis buffer (7.08 g 10 ml−1 guanidine thiocyanate [6M], made up to 8.2 ml with water, 1 ml Tris HCL [pH 8.0] and 800 µl of 1M dithiothreitol), mixed by pipetting ten times and incubation at room temperature for 1 min [26 (link)]. To this, 75 µl of 100% ethyl alcohol and 20 µl of prepared SPRI beads were added, mixed by pipetting ten times and incubated at room temperature for 5 min. The RNA-bead complex was then immobilized by placing the 96-well plate on a magnetic rack and incubated again at room temperature for 5 min. The supernatant was then discarded, and the beads washed twice in 200 µl of freshly prepared 80% ethyl alcohol (v/v) with 30 s room-temperature incubation between each wash. The beads were air-dried for 2 min at room temperature before RNA was eluted by the addition of 20 µl of nuclease-free water.
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