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Vial / Bottle | Cap | Label | Contents / Function |
---|
1 | green | Binding Buffer | - 2 × 25 ml
- [6 M guanidine-HCl, 10 mM Tris-HCl, 10 mM urea, 20% Triton X-100 (w/v), pH 4.4 (+25°C)]
|
2 | | Poly(A) | - Lyophilizate
- 2 mg poly(A) carrier RNA
- For binding of RNA
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3 | pink | Proteinase K | - Lyophilizate
- 100 mg
- For the digestion of proteins
|
4 a | black | Inhibitor Removal Buffer | - 33 ml, add 20 ml absolute ethanol
- [5 M guanidine-HCl, 20 mM Tris-HCl, pH 6.6 (+25°C) final concentration after addition of ethanol]
|
4 b | blue | Wash Buffer | - 2 × 10 ml add 40 ml absolute ethanol each
- [20 mM NaCl, 2 mM Tris-HCl, pH 7.5 (+25°C) final concentrations after addition of ethanol]
|
5 | colorless | Elution Buffer | |
6 | | High Pure Filter Tubes | - Two bags with 50 polypropylene tubes with two layers of glass fiber fleece, for use of up to 700 μl sample volume.
|
7 | | Collection Tubes | - Eight bags with 50 polypropylene tubes (2 ml).
|
All solutions are clear, except Vial 1 Binding Buffer is clear to slightly turbid, and colorless to slightly yellowish viscous solution and should not be used if precipitates are present. Warm the solutions at +15 to +25°C or in a +37°C waterbath until the precipitates are dissolved.
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Virus lysis is accomplished by incubation of the sample in a special Lysis/Binding buffer in the presence of Proteinase K. Subsequently, nucleic acids bind specifically to the surface of glass fibers in the presence of a chaotropic salt (1). The binding reaction occurs within seconds due to the disruption of the organized structure of water molecules and the interaction of nucleic acids with the glass fibers surface. Thus, adsorption to the glass fiber fleece is favored. Since the binding process is specific for nucleic acids, the bound nucleic acids are purified from salts, proteins and other impurities by a washing step and are eluted in low salt buffer or water.
- Serum, plasma or whole blood are lysed by incubation with Binding buffer and Proteinase K.
- Nucleic acids are bound to the glass fibers pre-packed in the High Pure Filter Tube.
- Bound nucleic acids are washed with a special Inhibitor Removal Buffer to get rid of PCR inhibitory contaminants. It allows even the application of heparinized sample material with > 100 U/ml heparin.
- Washing of bound nucleic acids, purification from salts, proteins and other cellular impurities.
- Purified nucleic acids are recovered using the Elution Buffer.
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Total time | Approximately 20 min |
---|
Hands-on time | <10 min |
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