Telomere length was measured by Southern blot previously described [26 (link)] and was conducted at RERF. In brief, genomic DNA was isolated from leukocytes using a DNA isolation kit (Qiagen) and digested with HinfI and RsaI (NEB). Digested DNA was separated on a 0.6% agarose gel by electrophoresis. The hybridization was performed with a [32 P] end-labeled oligonucleotide (TTAGGG)4 probe, at 45°C overnight. The images were acquired by a phosphor imager (Typhoon 9410; GE Biosciences). Mean telomere length (terminal restriction fragment, TRF) was calculated at the NIA in an observer blind manner.
Hinfi and rsai
Manufactured by New England Biolabs
HinfI and RsaI are type II restriction endonucleases that recognize and cleave specific DNA sequences. HinfI recognizes and cleaves the palindromic DNA sequence 5'-G^ANTC-3', while RsaI recognizes and cleaves the palindromic DNA sequence 5'-GT^AC-3'. These enzymes are commonly used in molecular biology applications, such as DNA digestion and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Typhoon biomolecular imager, by GE Healthcare (2 mentions)
Cutsmart buffer, by New England Biolabs (2 mentions)
Dna isolation kit, by Qiagen (1 mentions)
Typhoon 9410, by GE Healthcare (1 mentions)
Chef drii apparatus, by Bio-Rad (1 mentions)
Gentra puregen dna extraction kit, by Qiagen (1 mentions)
Hybond n membrane, by GE Healthcare (1 mentions)
Anti dig antibody, by Roche (1 mentions)
Cdp star, by Roche (1 mentions)
Dot blot apparatus, by Bio-Rad (1 mentions)
Hybond xl membrane, by Cytiva (1 mentions)
Phi29 dna polymerase, by New England Biolabs (1 mentions)
Hybond, by Cytiva (1 mentions)
4 protocols using hinfi and rsai
1
Telomere Length Measurement by Southern Blot
2
Telomeric DNA Restriction Analysis
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Genomic DNA (gDNA) was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and digested with HinfI and RsaI (NEB) in CutSmart buffer (NEB), overnight at 37°C. 0.5–2 μg of digested gDNA/lane were resolved by constant-field gel electrophoresis in 0.8% agarose gel in 1× TBE for 17 h at 70 V, or by pulsed-field gel electrophoresis in 1% agarose gel in 0.5x TBE for 16 h at 14°C, at 5.2 V/cm with 0.5 s initial switch and 6 s final switch, using a CHEF-DRII apparatus (Bio-Rad). The gel was dried for 4–5 h at 50°C, denatured in 0.5 M NaOH, 1.5 M NaCl for 15 min on a shaker at room temperature, neutralized in 0.5 M Tris-Cl pH 7.0, 1.5 M NaCl for 10 min on a shaker at room temperature, and then blocked in Church buffer (as described above) for at least 1 h at 50°C. The gel was hybridized with a 32P-radiolabeled TeloC probe in Church buffer at 50°C overnight. Washes were done for 1 h/wash at 50°C, in 4× SSC, followed by 4× SSC 0.1% SDS and 2× SSC 0.1% SDS. The gel was then exposed to a phosphorimager screen. Radioactive signal was detected with a Typhoon Biomolecular Imager (GE).
3
Telomere Length Quantification Protocol
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Genomic DNA was extracted by Gentra Puregen DNA Extraction Kit (Qiagen). DNA (3 μg) was digested with HinF I and Rsa I (NEB) overnight and separated on a 0.85% agarose gel at 2 V/cm for 16 hr. DNA was transferred from gel to Hybond‐N+ membrane (GE) and fixed by UV crosslinking. The membrane was hybridized with DIG‐labeled telomere probe overnight at 42 °C, followed by washing with 2× SSC, 0.1% SDS buffer for 15 min, 0.5× SSC, and 0.1% SDS buffer at 55 °C for 15 min twice. The membrane was incubated with 1× DIG blocking solution and then with anti‐DIG antibody (Roche) for 30 min. Telomere signals were detected by incubating with CDP‐star (Roche) for 5 min. The average telomere length was quantified using telotool software.
4
Quantifying Telomeric DNA using C-circle Assay
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The C-circle assay was performed as previously described (46 (link)). Briefly, genomic DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and digested with HinfI and RsaI (NEB) in CutSmart buffer (NEB), overnight at 37°C. 30 ng of digested DNA were incubated with 7.5 U Phi29 DNA polymerase (NEB) (or in its absence for Phi29− control reaction), in the presence of dATP, dTTP and dGTP (1 mM each) at 30°C for 8 h, followed by heat-inactivation at 65°C for 20 min. Reaction products were blotted onto a Hybond-XL membrane (Amersham) using a dot blot apparatus (Bio-Rad), and DNA was UV-crosslinked to the membrane. The non-denatured membrane was blocked in Church buffer (described above) for at least 1 h at 42°C. The membrane was hybridized with a 32P-radiolabeled TeloC oligonucleotide probe in Church buffer at 42°C overnight. Washes were done three times for 30 min/wash at 42°C, in 1× SSC, 0.5% SDS. The membrane was then exposed to a phosphorimager screen. Radioactive signal was detected with a Typhoon Biomolecular Imager (GE). For detection of the Phi29− signal, the membrane was stripped and denatured as mentioned above, blocked in Church buffer for at least 1 h at 42°C, and re-probed with a 32P-radiolabeled TeloC oligonucleotide probe in Church buffer at 42°C overnight. Washes and signal detection were done as described.
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