For detection of replication activities, each 10 μl extract sample was incubated with 2 μM of Cy5-dUTP (GE Healthcare) for the appropriate number of times and was diluted with 200 μl of stop buffer (5-mM EDTA, 200-mM NaCl, 0.5% SDS, 20-mM Tris-HCl, pH 8.0) containing 200 μg/ml Proteinase K (Roche) and 10 μg/ml RNaseA (Sigma-Aldrich) and incubated for 2 h at 37 °C. The genomic DNA was purified by phenol/chloroform extraction and ethanol precipitation and electrophoretically separated by 0.8% tris-acetate EDTA (TAE) agarose gel (neutral condition) or 1% alkaline agarose gel (denaturing condition) as described (50 (link)) and stained with SYBR Gold (Invitrogen). The alkaline gel was neutralized with PBS before SYBR Gold staining because it is pH sensitive. The signals of incorporated Cy5 (Replication activity) and SYBR Gold (total genomic DNA) were scanned with Typhoon FLA 9000 Gel Imager (GE Healthcare) and quantified with ImageJ software.
Typhoon fla 9000 gel imager
Manufactured by GE Healthcare
Sourced in Sweden
The Typhoon FLA 9000 gel imager is a high-performance fluorescence imaging system designed for the analysis of gels, blots, and other fluorescent samples. It uses a laser-based detection system to capture images with high sensitivity and resolution.
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4 protocols using typhoon fla 9000 gel imager
1
Quantifying DNA Replication Dynamics
2
Kinase Activity Assay Protocol
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Kinase assays were conducted in 30-µl reactions containing 20 mM Tris–HCl (pH 7.5), 5 mM EGTA, 1 mM dithiothreitol, 100 mM NaCl, 10 mM MgCl2, 100 µM [γ-32P]ATP mix (5 µCi of ATP; ATP, [γ-32P]-3000 Ci mmol−1, 10 mCi ml−1; EasyTide, 100 µCi), 10 μg of substrate proteins and 1 μg of kinases.
The reactions were incubated at 30 °C for 30 minutes and stopped with the SDS sample buffer. After SDS–PAGE, gels were dried, exposed to a GE multipurpose standard screen (catalogue no. 63-0034-87) for 18 h and imaged using a GE Typhoon FLA 9000 gel imager.
The reactions were incubated at 30 °C for 30 minutes and stopped with the SDS sample buffer. After SDS–PAGE, gels were dried, exposed to a GE multipurpose standard screen (catalogue no. 63-0034-87) for 18 h and imaged using a GE Typhoon FLA 9000 gel imager.
3
Western Blot and Fluorescence Imaging
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The density values for the Western blot fragments and immunofluorescence signals were analyzed by ImageJ (NIH freeware, Bethesda, MD, USA). We also measured the relative fluorescence intensity in the microirradiated ROI. The ROI data were normalized, and the fluorescence intensity measured outside the microirradiated regions. Scanning of the Western blot fragments on fluorescent gels was performed by a GE Typhoon FLA 9000 gel imager and ImageQuant TL 8.2 software (GE Healthcare, Uppsala, Sweden). The collected data from the Western blots were normalized to standards, including the level of the total histone H3 or α-tubulin. Sigma Plot software was used for the statistical analysis by Student’s t-test. In each experimental event, we analyzed 50–80 cell nuclei in three independent experiments. We used an online tool http://shiny.chemgrid.org/boxplotr/ for data plotting.
4
In Vitro Transcription and Radioactive Labeling of Pre-miR-20b RNA
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