supplementary_les_2

supplementary_les_2 - Supplementary information on reporter...

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Supplementary information on reporter genes 1 Supplementary information on reporter genes 1. CHLORAMPHENICOL ACETYL TRANSFERASE (CAT) Principle: (Naylor, Biochemical pharmacology, 1999) Reaction: CAM + Ac-CoA 3-Ac-CAM 1-Ac-CAM 1-Ac-CAM + Ac-CoA 1,3-diAc-CAM
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Supplementary information on reporter genes 2 Applications: Example 1: (Jin et al., Oncogene 2000)
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Supplementary information on reporter genes 3 Example 2: Figure 2. Effect of MEKinhibitor U0126 on viral gene expression. Cells are transfected with a reporterconstruct in which a viral gene is replaced by CATgene, but maintaining the viral 3’ and 5’ UTR. The effect of the MEK inhibitor U0126 (DMSO=control) on CAT activity is measured. Cai et al., J Virol 2007
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Supplementary information on reporter genes 4 2. β -GALACTOSIDASE (lactase) Principle:
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Supplementary information on reporter genes 5 www.appliedbiosystems.com
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Supplementary information on reporter genes 6 Description X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) is an inert chromogenic substrate for beta-galactosidase, an enzyme that promotes lactose utilization. Beta-galactosidase hydrolyzes X-Gal into a colorless galactose and 4-chloro-3-brom- indigo which forms an intense blue precipitate. Induction of the lacZ gene with IPTG leads to the hydrolysis of X-Gal and to the development of blue colonies ( see the scheme below). Applications Blue/white colony screening assay, distinguishing recombinant colonies (white) among non-recombinant ones (blue) ( 1 ), see Protocol for the Blue/White Colony Screening . Visualization of expression of the beta-galactosidase reporter gene in transfected eukaryotic cells, see Protocol for the Detection of the Beta-galactosidase Reporter Gene in Transfected Eukaryotic Cells . Detection of beta-galactosidase activity in immunological and histochemical procedures. Quality Control Greater than 98% purity confirmed by HPLC. Functionally tested in blue/white colony screening. Note Preparation of a 20 mg/ml stock solution in dimethylformamide or dimethylsulfoxide is recommended. Dimethylformamide dissolves some plastic materials. The direct addition of dimethylformamide containing solution to plastic Petri dishes should be avoided. Storage Store at -20°C in the dark . www.fermentas.com
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Supplementary information on reporter genes 7 Applications: Example 1: Protocol for the Blue/White Colony Screening For individual LB (Luria Broth) agar plates: 1. Pour sterile warm LB agar (about 25 ml) into a Petri dish. 2. Dry opened LB plates at room temperature under UV light for about 30 min. 3. Add 40 μl of the X-Gal Solution (20 mg/ml), ready-to-use. 4. Add 40 μl of 100 mM IPTG Solution, ready-to-use . 5.
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supplementary_les_2 - Supplementary information on reporter...

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