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[1.] Iam/Fragment 038 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:03:42 Graf Isolan
Fragment, Gesichtet, Iam, SMWFragment, Schutzlevel sysop, Sheikh 2006, Verschleierung

Typus
Verschleierung
Bearbeiter
Graf Isolan
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 38, Zeilen: 1ff (complete)
Quelle: Sheikh 2006
Seite(n): 35, 36, Zeilen: 35:4-25; 36:1ff
[The samples were centrifuged] overnight (21 h) at 35,000 rpm in a Kontron TST55 rotor at 20°C. The supernatants were carefully removed by aspiration and the transparent gelatin-like RNA pellets were gently washed (preserving undisturbed) with 200 μl of 70% ethanol at room temperature. The pellets were reconstituted in 200 μl of RNase-free water by pipetting and transferred into sterile 1.5 ml eppendorf tubes and the procedure was immediately continued to RNA precipitation.

RNA precipitation: The RNA was precipitated with 450 μl of 100% ethanol in the presence of sodium acetate, pH 5.4 (20 μl of 2 M solution per pellet) overnight at –20°C. The RNA precipitates were centrifuged for 30 min at 12,000 rpm in an Eppendorf bench-top centrifuge at 4°C to get RNA pellet.

Washing of the RNA pellet: Supernatants were discarded and pellet was washed with 200 μl of ice-cold 70% ethanol to remove all traces of sodium acetate. The RNA precipitates were centrifuged as described above, the supernatants were discarded and the pellet was dried for 30 min. at room temperature.

Reconstitution of RNA: The pellets were reconstituted in 100 μl of RNase-free water. To determine the concentration and purity of the RNA obtained, the aliquot of RNA sample was diluted 1:100 in RNase-free H2O and the concentration was measured at 260 nm and 280 nm by spectrophotometer (GeneQuant II, Pharmacia Biotech).

Solutions used for Ultracentrifugation

Guanidine isothiocyanate (GITC) buffer

    Final concentration
Guanidine isothiocyanate   4 M
0.25 M sodium citrate   25 mM
N-lauroylsarcosyl   0.5%

The solution was sterile filtered and stored in the dark at 4°C. β-Mercaptoethanol was added just prior to use at a ration of 1 to 100 μl of GITC buffer.

Cesium chloride (CsCl) buffer

    Final concentration
Cesium chloride   5.7 M
0.25 M sodium citrate   25 mM
0.5 M EDTA   100 mM

pH was adjusted with 0.25 M citric acid to 7.5; the solution was dissolved in RNase-free [H2O, sterile filtered and stored at room temperature.]

[Page 35]

The samples were centrifuged overnight (21 h) at 35,000 rpm in a Kontron TST55 rotor at 20°C. The supernatants were carefully removed by aspiration and the transparent gelatin-like RNA pellets were gently washed (preserving undisturbed) with 200 μl of 70% ethanol at room temperature. The pellets were reconstituted in 200 μl of RNase-free water by pipetting and transferred into sterile 1.5 ml eppendorf tubes and the procedure was immediately continued to RNA precipitation.

RNA precipitation

The RNA was precipitated with 450 μl of 100% ethanol in the presence of sodium acetate, pH 5.4 (20 μl of 2 M solution per pellet) overnight at –20°C. The RNA precipitates were centrifuged for 30 min at 12,000 rpm in an Eppendorf bench-top centrifuge at 4°C to get RNA pellet.

Washing of the RNA pellet

Supernatants were discarded and pellets were washed with 200 μl of ice-cold 70% ethanol to remove all traces of sodium acetate. The RNA precipitates were centrifuged as described above, the supernatants were discarded and the pellets were dried for 30 minutes at room temperature.

Reconstitution of RNA

The pellets were reconstituted in 100 μl of RNase-free water. To determine the concentration and purity of the RNA obtained, the aliquot of RNA sample was diluted 1:100 in RNase-free H2O and the concentration was measured at 260 nm and 280 nm by spectrophotometer (GeneQuant II, Pharmacia Biotech).

[Page 36]

Solutions used for Ultracentrifugation

Guanidine isothiocyanate (GITC) buffer
  For 200 ml Final concentration
Guanidine isothiocyanate 94.53 g 4 M
0.25 M sodium citrate 20 ml 25 mM
N-lauroylsarcosyl 1 g 0.5%
RNase-free H2O to 200 ml  

The solution was sterile filtered and stored in the dark at 4°C. β-Mercaptoethanol was added just prior to use at a ration of 1 to 100 μl of GITC buffer.

Cesium chloride (CsCl) buffer
  For 200 ml Final concentration
Cesium chloride 192 g 5.7 M
0.25 M sodium citrate 20 ml 25 mM
0.5 M EDTA 40 ml 100 mM
RNase-free H2O to 200 ml  

pH was adjusted with 0.25 M citric acid to 7.5; the solution was sterile filtered and stored at room temperature.

Anmerkungen

Nearly identical, nevertheless not marked as a citation.

Sichter
(Graf Isolan) Schumann


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