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[1.] Iam/Fragment 039 01 - Diskussion
Zuletzt bearbeitet: 2014-03-12 20:16:56 Graf Isolan
Fragment, Gesichtet, Iam, KomplettPlagiat, SMWFragment, Schutzlevel sysop, Sheikh 2006

Typus
KomplettPlagiat
Bearbeiter
Graf Isolan
Gesichtet
Yes.png
Untersuchte Arbeit:
Seite: 39, Zeilen: 1ff (complete)
Quelle: Sheikh 2006
Seite(n): 36-37, 38, Zeilen: 36:16-28 - 37:1-6.16-26; 38:2-4.8-10
[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.

3.2.4 Northern blot analysis

Northern blot analysis is a method to quantify RNA expression. The RNA is separated in a denaturing formaldehyde/agarose gel, transferred by capillary transfer to a nylon membrane and fixed by UV cross linking. The RNA of interest is identified by hybridization with a specific radiolabeled cDNA probe. All solutions used for the northern blot were autoclaved, the electrophoresis and blot chambers, gel plates and combs were kept in freshly prepared 0.1% DEPC solution for 10-20 min before use to inactivate RNases.

3.2.4.a. Preparation of RNA samples

Each RNA probe (5-10 μg of total RNA) in a volume not more than 5 μl was mixed with 7.5 μl of sample buffer. In case of dilute sample where the volume of the probe exceeded 5 μl, the sample was concentrated in a vacuum centrifuge until the volume was reduced to 5 μl. RNA probes mixed with sample buffer were denatured by heating at 65°C for 10 min. Afterwards, the samples were briefly cooled on ice and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge. Each sample was mixed with 3 μl of loading buffer and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge.

3.2.4.b. Electrophoresis

The electrophoresis was performed at constant voltage of 80 V for 1-1.5 h. After electrophoresis, the quality of RNA was estimated under UV transilluminator built-in Eagle Eye™ system (Stratagene); the gel was photographed and the procedure was immediately continued to blotting.

3.2.4.c. Transfer of RNA to nylon membrane

After the separation of RNA in the gel, it was transferred to a nylon membrane by capillary transfer method. A plastic tray was filled with 500 ml of 20X SSC and a piece of Whatman 3MM filter was soaked in 20X SSC and swaddled over the glass plate placed over the tray with both ends hanging into buffer to act as wick. The transfer was carried out overnight. After the transfer, RNA was fixed on the membrane by UV cross linking for 2 min from both sides using Stratalinker™ 180 (Stratagene) set at “autocross link” mode.

Pre-hybridization: Afterwards, the membrane was placed into a hybridization tube and any bubbles between the membrane and internal wall of the tube were carefully removed.

[Page 36]

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

3.2.3 Northern blot analysis

Northern blot analysis is a method to quantify RNA expression. The RNA is separated in a denaturing formaldehyde/agarose gel, transferred by capillary transfer to a nylon membrane and fixed by UV cross linking. The RNA of interest is identified by hybridization with a specific radiolabeled cDNA probe. All solutions used for the northern blot were autoclaved, the electrophoresis and blot chambers, gel plates and combs were kept in freshly prepared 0.1% DEPC solution for 10-20 min before use to inactivate RNases.

3.2.3.I Preparation of RNA samples

Each RNA probe (5-10 μg of total RNA) in a volume not more than 5 μl was mixed with 7.5 μl of sample buffer. In case of dilute sample where the volume of the

[Page 37]

probe exceeded 5 μl, the sample was concentrated in a vacuum centrifuge until the volume was reduced to 5 μl. RNA probes mixed with sample buffer were denatured by heating at 65°C for 10 min. Afterwards, the samples were briefly cooled on ice and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge. Each sample was mixed with 3 μl of loading buffer and centrifuged for 1 min at 10,000 rpm in an Eppendorf bench-top centrifuge.

[...]

3.2.3.III Electrophoresis

The electrophoresis was performed at constant voltage of 80 V for 1-1.5 h. After electrophoresis, the quality of RNA was estimated under UV transilluminator built-in Eagle Eye™ system (Stratagene); the gel was photographed, and the procedure was immediately continued to blotting.

3.2.3.IV Transfer of RNA to nylon membrane

After the separation of RNA in the gel, it was transferred to a nylon membrane by capillary transfer method. The system for transfer was assembled as depicted in Figure 4. A plastic tray was filled with 500 ml of 20×SSC. A piece of Whatman 3MM filter was soaked in 20×SSC and swaddled over the glass plate placed over the tray with both ends hanging into buffer to act as wick. [Any bubbles between the filter and glass plate were smoothed out. The gel was carefully placed upside down over the filter. The bubbles between the gel and wick were carefully removed. The nylon membrane wetted in 2×SSC was placed on top of the gel and smoothed. Two other pieces of Whatman 3MM filters soaked in 2×SSC were sequentially flat laid on top of nylon filter and smoothed. A stack]

[Page 38]

[of paper towels was placed on top, covered with another glass plate and pressed with 1 kg blotting weight.] The transfer was carried out overnight. After the transfer, RNA was fixed on the membrane by UV cross linking for 2 min from both sides using Stratalinker™ 180 (Stratagene) set at “autocross link” mode.

[...]

Pre-hybridization

Afterwards, the membrane was placed into a hybridization tube, and any bubbles between the membrane and internal wall of the tube were carefully removed.

Anmerkungen

Identical, without a hint that Iam is not the author of this text.

In "3.2.4.c." Iam has left out some of the original text, thus in fact the description of the procedure is incomplete.

Sichter
(Graf Isolan) Schumann


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