The seeds of IR64 (International Rice Genebank Collection #66970, selfed for at least 10 times at National Institute of Agrobiological Sciences, Japan) were sterilized and incubated on Murashige and Skoog medium supplemented with 3% sucrose and 1% agar at pH 5.8 in a plant box at 28° for 8 days. Leaves from the 8-day-old seedlings were frozen in liquid nitrogen and ground to a fine powder with a mortar and pestle. High-molecular-weight DNA was extracted with Buffer G2 (Qiagen) supplemented with proteinase K and RNase A at 60 ° overnight with gentle agitation. After centrifugation at 2000 ×g for 30 min, the supernatant was loaded to a genomic-tip 100 (Qiagen) pre-equilibrated with Buffer QBT (Qiagen) and washed with Buffer QC (Qiagen) twice. DNA was eluted with Buffer QF (Qiagen), precipitated with isopropyl alcohol, washed with 70% ethanol, and dissolved in Buffer EB (Qiagen). The concentration of DNA was measured with the Qubit dsDNA High Sensitivity Assay Kit (Invitrogen).
Buffer qbt
Manufactured by Qiagen
Sourced in Germany
Buffer QBT is a buffer solution used in nucleic acid purification protocols. It is designed to provide optimal binding conditions for the adsorption of nucleic acids to a solid support matrix, such as silica membranes or resins. The exact composition and formulation of Buffer QBT are proprietary information.
Automatically generated - may contain errors
Lab products found in correlation
Genomic tip kit, by Qiagen (2 mentions)
Miseq sequencer, by Illumina (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Buffer g2, by Qiagen (1 mentions)
Eb buffer, by Qiagen (1 mentions)
Genomictip100, by Qiagen (1 mentions)
Buffer qf, by Qiagen (1 mentions)
Qubit dsdna high sensitivity assay kit, by Thermo Fisher Scientific (1 mentions)
Lobind tube, by Eppendorf (1 mentions)
Falcon tube, by BD (1 mentions)
4 protocols using buffer qbt
1
High-Quality DNA Extraction from Rice Seedlings
2
Genomic DNA Extraction Protocol
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This protocol uses ordinary pipettes (max volume: 20 µl, 200 µl, 1 ml) from Gilson (Middleton, WI, USA) or Rainin™ (Metter-Toledo Rainin, Oakland, CA, USA) and respective pipette tips for general pipetting, while for the lysate and steps involving DNA, wide-bore 1 ml pipette tips (Metter-Toledo Rainin) were used. Falcon tubes (50 ml), Eppendorf tubes (2 ml, Eppendorf, Hamburg, Germany), and Eppendorf LoBind tubes (1.5 ml) were used throughout the protocol. Corning® Cell Strainers (Corning Inc. Corning NY, USA) of 100 µm pore size were used for small scale extractions. Qiagen Genomic-tip 20/G and 100/G (#10223, #10243, Qiagen, Hilden, Germany) columns were used in combination with the modified CTAB buffer described above, and buffer QBT, buffer QC, and buffer QF as purchased from Qiagen. Hydrogen-ion exponent (pH) indicating strips (Fisherbrand™ pH 4.5–10; Thermo Fisher Scientific, Waltham, MA, USA) were used to check the pH of the lysate.
3
Polysaccharide-rich DNA Extraction from D. molle
4
Polysaccharide-rich DNA Extraction from D. molle
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Extraction of DNA from D. molle samples E11-036 and E11-037 was complicated by the presence of copious amounts of polysaccharide-rich mucus. We utilized a protocol previously adapted from Sokolov for the precipitation of polysaccharides found in marine invertebrate slime.51 ,52 (link) Briefly, tissue was sliced thinly and squashed between two aluminum foil sheets, then treated with proteinase K (Qiagen). Polysaccharides and proteins were then precipitated with saturated KCl. Additional KCl was required beyond what was called for in the original protocol. Samples were centrifuged and an equal volume of isopropanol added to the supernatant. White precipitate formed upon incubation at room temperature. The mixture was centrifuged again and the pellet washed with 70% ethanol. Excess solvent was removed by air drying and the pellet resuspended in QBT buffer (Qiagen). From this point forward, DNA extraction followed the standard protocol for the Qiagen Genomic-tip kit. An Illumina library was prepared using the E11-036 DNA with ~700 bp insert sizes and then sequenced on an Illumina MiSeq sequencer using a 250 bp paired end run. For D. molle sample E11-037, a 350 bp insert size library was prepared and sequenced (100 bp paired end) on an Illumina HiSeq 2500.
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