Genomic DNA was extracted using the Qiagen DNeasy Kit (Qiagen) according to the manufacturer’s instructions. A total of 5 μg of DNA was digested overnight with HinfI and RsaI (40 U each). The digested DNA was resolved on a 1% SeaKem Gold agarose gel (Lonza) in 0.5×Tris/Borate/EDTA (TBE) buffer, using a CHEF electrophoresis chamber (BioRad). Electrophoresis was conducted with initial switch time 0.2 s and final switch time 13 s at 6 V/cm for 15 h. The gel was blotted onto a positively charged Biodyne B nylon membrane (Pall, VWR) and hybridized at 42°C in 6 × saline-sodium citrate buffer (SSC), 0.01% sodium dodecyl sulphate (SDS) with 20 pmol of digoxigenin (DIG)-labeled telomeric C-rich oligonucleotide TAA(CCCTAA)4 prepared using the 3′ end labeling kit (Roche, 03353575910). Following hybridization washes (twice 5 min in 2 × SSC, 0.01% SDS and once 2 min in 0.1 × SSC, 0.01% SDS) the signal was revealed using the anti-DIG-alkaline phosphatase antibodies (Roche, 11093274910) and CDP-Star (Roche) following the manufacturer’s instructions. Images were obtained using the Luminescent image analyzer LAS-4000 mini (GE Healthcare) and analyzed using the Telometric 1.2 software.
3 end labeling kit
Manufactured by Roche
The 3' end labeling kit is a laboratory product designed for the labeling of nucleic acid sequences at the 3' end. It provides the necessary reagents and protocols for the efficient and precise labeling of DNA or RNA fragments, enabling researchers to conduct various downstream applications that require labeled nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Biodyne b nylon membrane, by Avantor (2 mentions)
Las 4000 mini, by GE Healthcare (2 mentions)
Cdp star, by Roche (2 mentions)
Seakem gold agarose gel, by Lonza (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
γ 32p datp, by PerkinElmer (1 mentions)
Anti digoxigenin alkaline phosphatase antibody, by Roche (1 mentions)
2 protocols using 3 end labeling kit
1
Telomeric DNA Analysis by Pulsed-Field Gel Electrophoresis
2
Chromatin Immunoprecipitation Telomere Analysis
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ChIP was performed using standard procedures and antibodies listed in Supplementary Table S1 . A detailed protocol is provided in Supplementary Methods . Purified DNA recovered by ChIP was denatured in 0.2 M NaOH by heating to 100°C for 10 min and spotted onto a positively charged Biodyne B nylon membrane (Pall, VWR) before hybridization with a digoxigenin-labeled telomeric C-rich oligonucleotide (CCCTAA)4TTA prepared using 3′-end labeling kit (Roche). Signal was revealed using the anti-digoxigenin-alkaline phosphatase antibodies (Roche) and CDP-Star (Roche), and images were captured using the Luminescent image analyzer LAS-4000 mini (GE Healthcare). Centromeric probe with sequence: 5′-CTTCGTTGGAAACGGGA (forward strand of the CenP Box) was used as a control. The primer was polyacrylamide gel electrophoresis purified and end-labeled with [γ32P]dATP (PerkinElmer) using PNK followed by purification on G-25 column.
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