Images of agarose gels were acquired on an Alpha Innotech AlphaImager 2200. Chemiluminescent immunoblots and methylene blue–stained RNA blots were imaged with a Bio-Rad ChemiDoc, and radiolabeled RNA blots were imaged with an Amersham Typhoon IP Biomolecular Imager. Image quantification of gels and blots was conducted with ImageJ version 1.51j8. All quantifications were made using unaltered, uncompressed original image files. Entire images presented in the manuscript may be adjusted for lightness or contrast for better clarity and consistency. No data were obscured by such processing. All data analysis was conducted on unaltered raw images.
Typhoon ip biomolecular imager
Manufactured by Cytiva
The Typhoon IP Biomolecular Imager is a high-performance imaging system designed for analyzing and quantifying a variety of biomolecules, including proteins, nucleic acids, and small molecules. The system utilizes a range of excitation and emission wavelengths to detect and capture images of fluorescently labeled samples.
Automatically generated - may contain errors
2 protocols using typhoon ip biomolecular imager
1
Imaging and Quantification of Biomolecular Blots
2
Quantitative Telomere Analysis by Southern Blot
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Cells (3-5 x10 8 ) were collected at each time point and used to prepare genomic DNA as in (41) .
Genomic DNA (50 µg) was digested with 20 units of Sca I-HF (NEB) overnight at 37°C. The digested DNA was resolved on a 1.2% agarose gel (6 h at 100 V) and transferred onto a Hybond N+ positively charged nylon membrane (Amersham -GE healthcare). The membrane then was crosslinked with UV and hybridized (60°C, overnight) by labeled probes produced by PCR with u4ScaProbe_S + u4ScaProbe_AS (ura4 + probe), SV40ScaProbe_S + SV40ScaProbe_AS (hph + probe), or abo1-probe_S + abo1-probe_AS (abo1 + probe as a loading control) using 100 µCi of [α-32 P-dCTP] (6000 Ci/mmol, PerkinElmer) as previously described in (31) (primer sequences are shown in Tables 5). The labeled and washed membrane was exposed 1-2 days into a cassette containing a phosphor screen. The radiolabeled DNA quantification acquisition was made using the Amersham Typhoon IP Biomolecular Imager. All p. 8 of 43 the quantifications and telomere band size measurements were then made using Image Quant TM TL 8.2 Software for Windows.
Genomic DNA (50 µg) was digested with 20 units of Sca I-HF (NEB) overnight at 37°C. The digested DNA was resolved on a 1.2% agarose gel (6 h at 100 V) and transferred onto a Hybond N+ positively charged nylon membrane (Amersham -GE healthcare). The membrane then was crosslinked with UV and hybridized (60°C, overnight) by labeled probes produced by PCR with u4ScaProbe_S + u4ScaProbe_AS (ura4 + probe), SV40ScaProbe_S + SV40ScaProbe_AS (hph + probe), or abo1-probe_S + abo1-probe_AS (abo1 + probe as a loading control) using 100 µCi of [α-32 P-dCTP] (6000 Ci/mmol, PerkinElmer) as previously described in (31) (primer sequences are shown in Tables 5). The labeled and washed membrane was exposed 1-2 days into a cassette containing a phosphor screen. The radiolabeled DNA quantification acquisition was made using the Amersham Typhoon IP Biomolecular Imager. All p. 8 of 43 the quantifications and telomere band size measurements were then made using Image Quant TM TL 8.2 Software for Windows.
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