Oligos (10 pmols for U6) were 5′-end labeled with [γ-32P]-ATP (PerkinElmer), using T4 PNK and 10X reaction buffer A (ThermoFisher Scientific) at 37°C for 1 h, then heat inactivated at 75°C for 10 min. The labeled oligos were then gel purified on a 19–20% PAGE denaturing gel (AccuGel 19:1, National Diagnostics), followed by extraction, ethanol precipitation, resuspension in buffer, and quantification by liquid scintillation counting.
Reaction buffer a
Manufactured by Thermo Fisher Scientific
Sourced in United States
10X reaction buffer A is a concentrated buffer solution used in various molecular biology and biochemistry applications. It provides the optimal ionic conditions for enzymatic reactions, such as DNA amplification or protein synthesis, when diluted to the recommended working concentration. The buffer composition ensures the proper function of the relevant enzymes or reagents during the experimental procedure.
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Lab products found in correlation
2 protocols using reaction buffer a
1
Radiolabeling and Purification of Oligos
2
Radioactive Primer Labeling Protocol
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For a total volume of 20 µL, the following contents were added to the mixture: 4 µL of reverse primer (CCTACGGTTACCTTGTT) with an initial concentration of 20 µM; 2 µL of 10X reaction Buffer A (ThermoFisher Scientific, Waltham, MA, USA); 2 µL of labeled γ-[32P] ATP; 10.2 µL of Milli-Q ultrapure water; and 1.8 µL of T4 Polynucleotide Kinase (Thermo Fisher Scientific, Waltham, MA, USA). The samples were then mixed, spun down and incubated for 30 min at 37 °C, followed by storage at −20 °C.
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