In molecular biology, double-stranded DNA probes are among the most commonly used tools for detecting and diagnosing transgenic plants. These probes are typically synthesized using two primary methods: nick translation and random primer synthesis. (1) 10× Nick Translation Buffer: 0.5 M Tris-Cl (pH 7.2), 0.1 M MgSOâ‚„, 10 mM DTT, and 100 µg/mL BSA. (1) Prepare the reaction mixture according to the following components: 1. While various isotopes such as ³H, ³²P, and ³âµS can be used for labeling, [α-³²P]-dNTPs are most commonly employed due to their high sensitivity and ease of detection. AD8 standard floating Solar Cable is a kind of equipment used for floating photovoltaic power generation. It consists of photovoltaic cells that convert solar energy into electricity. The device is designed to float on the water, while it can be fixed to the water by a buoy and anchorage device to prevent drifting. AD8 Floating Solar Cable,Solar Adapter Cable,Waterproof Solar Cable,DC Cable Suzhou Yonghao Cable Co.,Ltd. , https://www.yonghaocable.com Experimental Principle
Nick translation involves creating a single-strand break (nick) in one of the DNA strands. The E. coli DNA polymerase I then binds to the 3' hydroxyl end of the nick and begins synthesizing a new DNA strand by adding nucleotides. Simultaneously, the enzyme exhibits 5' to 3' exonuclease activity, which removes nucleotides from the 5' side of the nick. This process results in the migration of the nick along the DNA strand, allowing the replacement of non-radioactive nucleotides with radioactive ones, such as [α-³²P]dNTPs. The typical size of a nick translation fragment is between 50 and 500 nucleotides. Several factors influence the efficiency and outcome of the reaction: (a) The specific activity of the final product depends on both the specific activity of the [α-³²P]dNTP and the extent of template nucleotide replacement. (b) The concentration of DNase I and the amount of E. coli DNA polymerase I affect the size of the resulting fragments. (c) Contaminants like agarose in the DNA template can inhibit enzyme activity, so it's crucial to use purified DNA for optimal results. Experimental Reagents
(2) Unlabeled dNTP Stock Solution: A mixture of three unlabeled deoxynucleotides dissolved in 50 mM Tris·Cl (pH 7.5) at a concentration of 0.3 mM each.
(3) [α-³²P]dCTP or [α-³²P]dATP: Available at 400 Ci/mM and 10 µCi/µL.
(4) E. coli DNA Polymerase I: Concentrated at 4 units/µL, stored in 50 µg/mL BSA, 1 mM DTT, 50% glycerol, and 50 mM Tris·Cl (pH 7.5).
(5) DNase I: 1 mg/mL.
(6) EDTA: 200 mM (pH 8.0).
(7) 10 M Ammonium Acetate. Experimental Procedure
Add water to bring the total volume to 50 µL.
(2) Incubate the mixture in a 15°C water bath for 60 minutes.
(3) Stop the reaction by adding 5 µL of EDTA.
(4) Add ammonium acetate to achieve a final concentration of 0.5 M, then precipitate the DNA probe by adding two volumes of pre-chilled anhydrous ethanol. Precautions
2. The activity of DNase I significantly affects the quality of the probe. Higher DNase I activity leads to higher specific activity but shorter probe lengths. It’s important to optimize the DNase I concentration based on the desired fragment size and labeling efficiency.
3. Always handle radioactive materials with proper safety precautions and follow institutional guidelines for safe disposal.
4. Ensure that all reagents are freshly prepared and stored under appropriate conditions to maintain their activity and effectiveness.
5. For best results, perform the experiment in a clean and controlled environment to avoid contamination.
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