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| Untersuchte Arbeit: Seite: 25, Zeilen: 18-32 |
Quelle: Embark 2004 Seite(n): 45, Zeilen: 2ff |
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| 3.3.1 In vitro RNA transcription
In-vitro cRNA transcription involves 2 consecutive steps i.e. linearization of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA. a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight. b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose. c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA. d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm. |
2.2.1 In vitro cRNA transcription
As illustrated in Fig. 12, in vitro cRNA transcription involves 2 consecutive steps i.e. linearisation of the plasmid DNA containing the inserted cDNA of interest by the corresponding restriction enzyme and the synthesis of RNA. a. The inserted DNA should be cut at the 3’ end yielding a 5’ protruding or a blunt end by restriction enzyme. Plasmid DNA (10 μg) was incubated with 20 U restriction enzyme and an 10x buffer (5 μl) in a final volume of 50 μl at 37°C for 2 h or overnight. b. To ascertain the linearization process, a 5 μl aliquot was taken out and analysed on a 1% agarose. c. 1 volume isopropanol (50 μl) and 1/10 volume 3 M sodium acetate (5 μl) pH 5.2 was then added and incubated at room temperature for 10 min to precipitate the DNA. d. The precipitated DNA was recovered by centrifugation at 17,000 rpm for 15 min at 4°C. The DNA pellet was washed by adding 100 μl of cold 70% ethanol to the pellet followed by centrifugation at 17,000 rpm for 5 min at 4°C. This washing stage was repeated. The DNA pellet was air dried and then resuspended in 10 μl of DNase free H2O. The concentration of DNA was determined spectrophotometrically by measuring the absorbance at 260 nm. |
The source is not mentioned. It is not surprising that identical procedures are used in different studies and it makes sense to describe those procedures with the same words, but it should be made transparent. |
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