Telomeric probes 5′-(TTAGGG)4-3′ were radiolabeled by incubating 100 pmoles of each with 50 μCi of 32P-ATP and 50 units of T4 Polynucleotide Kinase (Thermo Fisher Scientific) in 50 μl of 1 × PNK buffer A for 1 h at 37 °C. The reactions were heat inactivated for 10 min at 75 °C and purified using the Micro Bio-Spin P-30 (Bio-Rad). The membranes were incubated for 30 min at 65 °C in Church Mix hybridization buffer (500mM NaPi pH 7.2, 1mM EDTA pH 8.0, 7% SDS, 1% BSA), and then overnight at 65°C with 10 ml of hybridization buffer containing the radiolabeled probe. The membranes were washed 4 times in 2X SSC at 65°C before exposure to a PhosphorImager screen and quantification with ImageJ software.
Micro bio spin p 30
Manufactured by Bio-Rad
The Micro Bio-Spin P-30 is a pre-packed size exclusion chromatography column designed for the rapid purification and desalting of small DNA, RNA, and protein samples. It is capable of separating molecules based on their size and molecular weight, allowing for the efficient removal of salts, small molecules, and other contaminants from the sample.
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Lab products found in correlation
2 protocols using micro bio spin p 30
1
Telomeric Probe Radiolabeling and Hybridization
2
Quantitative Telomeric DNA Analysis
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Telomeric probes 5′-(TTAGGG)4-3′ were radiolabeled by incubating 100 pmoles of each with 50 μCi of 32P-ATP and 50 units of T4 Polynucleotide Kinase (Thermo Fisher Scientific) in 50 μl of 1 × PNK buffer A for 1 h at 37 °C. The reactions were heat inactivated for 10 min at 75 °C and purified using the Micro Bio-Spin P-30 (Bio-Rad). The membranes were incubated for 30 min at 65 °C in Church Mix hybridization buffer (500mM NaPi pH 7.2, 1mM EDTA pH 8.0, 7% SDS, 1% BSA), and then overnight at 65°C with 10 ml of hybridization buffer containing the radiolabeled probe. The membranes were washed 4 times in 2X SSC at 65°C before exposure to a PhosphorImager screen and quantification with ImageJ software.
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