G1-arrested cells were released to S phase for 45 min and embedded into agarose plugs for PFGE analyses as previously described (Cremona et al., 2012 (link)). Briefly, plugs were treated with zymolyase (20T, MP Biomedicals), proteinase K, and lauroylsarcosine to permeabilize cells. Chromosomes were separated by 1% agarose (Bio-Rad) gels in 0.5× TBE buffer using the Bio-Rad CHEF-DR III PFGE system. Gel running conditions were 70–160 s switch time, 5.5 V/cm voltage gradient, and 106° angle for 15 h at 12°C. After electrophoresis, chromosomes were transferred to Hybond-XL nylon membranes (GE) for Southern blotting using an [α-32P]-dCTP-labeled rDNA probe. Autoradiographic signals were scanned by Typhoon FLA 9500 phosphoimager (GE), and rDNA replication efficiency was assessed by calculating the ratio of chromosome band signals to the corresponding well signals after adjusting for background signals. Quantification of chromosomal bands was performed using the ImageJ software.
Typhoon fla 9500 phosphoimager
Manufactured by GE Healthcare
Sourced in United Kingdom
The Typhoon FLA 9500 phosphoimager is a high-performance instrument designed for the detection and quantification of radiolabeled biomolecules in various applications, such as gel electrophoresis, membrane-based assays, and microarrays. The system utilizes a sensitive photomultiplier tube and a laser-based excitation source to enable high-resolution, high-sensitivity imaging of radioisotope-labeled samples.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant tl, by GE Healthcare (2 mentions)
γ 32p atp, by Hartmann Analytic (2 mentions)
Chef dr 3 pfge system, by Bio-Rad (1 mentions)
Hybond xl nylon membrane, by GE Healthcare (1 mentions)
Zymolyase 20t, by MP Biomedicals (1 mentions)
Tetrad2 thermocycler, by Bio-Rad (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
α 32p atp, by PerkinElmer (1 mentions)
Phi29 buffer, by New England Biolabs (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Amersham hybond n, by GE Healthcare (1 mentions)
Phi29 dna polymerase, by New England Biolabs (1 mentions)
Bas ms imaging plate, by Fujifilm (1 mentions)
Illustra microspin g 25 column, by GE Healthcare (1 mentions)
T4 pnk, by New England Biolabs (1 mentions)
10 protocols using typhoon fla 9500 phosphoimager
1
PFGE Analysis of rDNA Replication
2
Telomere Length Analysis by STELA
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DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Manchester, UK). For telomere length analysis at the XpYp telomere, we used the single telomere length analysis (STELA) assay, as previously described [39 (link),44 (link)], Genomic DNA was solubilized and diluted in 10 mmol/L Tric-HCl (pH 7.5) to 10 ng/µL. Ten nanograms of DNA were further diluted with 1 µmol/L Telorette2 linker and 1 mM Tris-HCl to 250 pg/µL in a volume of 40 µL. Multiple polymerase chain reactions were conducted to test the DNA sample and were cycled in a Tetrad2 thermocycler (BioRad, Hertfordshire, UK) (22 cycles of 94 °C for 15 s, 65 °C for 30 s, 68 °C for 8 min). DNA fragments were resolved by 0.5% TAE agarose gel electrophoresis and detected by southern hybridization. The hybridized fragments were detected using a phosphorimaging with a Typhoon FLA 9500 phosphoimager (GE healthcare, Chalfont St Giles, UK). The molecular weights of the DNA fragments were calculated using the Phoretix ID quantifier (Nonlinear Dynamics, Newcastle Upon Tyne, UK).
3
Radioactive and Fluorescent Gel Analysis
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All transcription reactions, except the reusability reactions shown in Figure 5 , were labeled with [α-32P]ATP (PerkinElmer). The gel showing reusability reactions in Figure 5 was stained with SYBR Gold Nucleic Acid Gel Stain (Invitrogen). All reactions were analyzed by 20% polyacrylamide (19:1 acrylamide:bisacrylamide) denaturing (7 M) urea gel electrophoresis. Gels labeled with radioactivity were dried and then imaged with a GE Typhoon FLA 9500 Phosphoimager. Gels labeled with SYBR Gold stain were imaged directly with a Bio-Rad Gel Doc Go Imaging System Blue Tray. Quantifications of gel lanes were performed using Fiji (63 (link)) software on raw TIF output.
4
Quantifying Telomeric C-Circles by Dot-Blot
5
Fluorescence-based Ubiquitination Assay
6
Fusion PCR for Inter- and Intra-Allelic Events
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Fusion PCR was undertaken as described [36 (link)]. Three primers were used to amplify inter- and intra- allelic fusion events in the HCA2 model: 17p6, 21q1 and XpYpM. The PCR conditions were as follows: 94°C for 20 s; 62°C for 30 s; and 68°C for 8 min and repeated for 25 cycles. DNA fragments were resolved with 0.5% TAE agarose gel electrophoresis and detected by Southern hybridisation at 55°C overnight with a 32P radiolabelled chromosome-specific (17p, 21q and XpYp) probe together with probes to detect the 1 kb and 2.5 kb markers. The blots were then washed four times at 55°C with 0.1% SDS and 0.1X SSC, dried, exposed to a phosphor screen and scanned with a Typhoon FLA 9500 phosphoimager (GE Healthcare) and analysed using ImageQuant TL (GE Healthcare).
7
Kinase Assay for QseE and QseF
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Kinase assays were performed in total volumes of 10–30 μl, depending on the number of aliquots to be analyzed over time. 1 μM QseE’‐His10 was pre‐incubated in reaction buffer (50 mM Tris–HCl, pH 7.6, 200 mM KCl, 10 mM MgCl2, 5 mM MnCl2) for 5 min at 25°C in the absence or presence of 5 μM Strep‐RapZ. For analysis of QseE’‐His10 autophosphorylation, 10 μCi [γ‐32P]‐ATP (Hartmann Analytic) and 100 μM cold ATP were added and aliquots were removed at indicated times. To analyze phosphoryl‐group transfer from QseE’‐His10 to QseF‐His10, QseE’‐His10 was incubated with [γ‐32P]‐ATP/ATP for 10 min and subsequently 1 μM QseF‐His10 was added. Aliquots were removed at indicated times and mixed with 2 × SDS sample buffer to stop reactions. The proteins were separated by SDS–PAGE and subsequently analyzed by using a Typhoon FLA‐9500 phospho‐imager (GE Healthcare).
8
Telomere Length Analysis in Cell Lines
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Single telomere length analysis (STELA) was undertaken as described [35 (link)]. Two primers were used to amplify specific telomeres in the HCA2 model: XpYpE2 and 17p6. Three primers were used to amplify specific telomeres in the MRC5 model: XpYpE2, XpYpAT and XpYpGC. Six primers were used to amplify specific telomeres in the HCT116 model: XpYpC, 5p5, 7qK1, 8q2, 9p2 and 17pseq1rev. The PCR conditions were as follows: 94°C for 20 s; 59°C (5p5, 17pseq1rev and 17p6), 61°C (9p2), 65°C (7qK1, 8q2, XpYpC, XpYpE2, XpYpAT and XpYpGC) for 30 s; 68°C for 8 min for 22 cycles. DNA fragments were resolved with 0.5% TAE agarose gel electrophoresis and detected by Southern hybridisation at 55°C overnight with a 32P radiolabelled telomere repeat (TTAGGG)n-containing probe together with probes to detect the 1 kb and 2.5 kb markers. The blots were washed four times at 55°C with 0.1% SDS and 0.1X SSC, dried, exposed to a phosphor screen and scanned with a Typhoon FLA 9500 phosphoimager (GE Healthcare) and analysed using ImageQuant TL (GE Healthcare).
9
APC/C-Mediated Ubiquitination Assay Protocol
10
Oligonucleotide Radiolabeling and Northern Blot
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The oligonucleotides used are listed in Supplementary Table 1. Oligonucleotides (150 pmoles) were 5' 32 P-labeled with 20 µCi of γ-32 P ATP (3000 Ci/mmole; Hartmann Analytic) for 30 min at 37°C in 50 µl volumes with 20 units of T4 PNK (New England Biolabs) in the provided reaction buffer. Reactions were heat inactivated for 20 min at 65°C and the unincorporated radioactivity was eliminated with a G25 Illustra MicroSpin column (GE Healthcare) according to the manufacture's instructions. Labeling efficiency was determined by comparing the radioactivity of the recovered material with that retained on the column.
Blots were incubated with 15 pmoles of probes overnight at 42°C. Blots were washed two times at 42°C with 2X SSC buffer (Euromedex, Souffelweyersheim, France) with 0.1% SDS added, washed two times with 0.2X SSC buffer with 0.1% SDS, dried and then subjected to autoradiography with a BAS-MS imaging plate (Fujifilm) overnight. Exposures were revealed with a Typhoon FLA9500 phosphoimager (GE Healthcare).
Blots were incubated with 15 pmoles of probes overnight at 42°C. Blots were washed two times at 42°C with 2X SSC buffer (Euromedex, Souffelweyersheim, France) with 0.1% SDS added, washed two times with 0.2X SSC buffer with 0.1% SDS, dried and then subjected to autoradiography with a BAS-MS imaging plate (Fujifilm) overnight. Exposures were revealed with a Typhoon FLA9500 phosphoimager (GE Healthcare).
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