A probe is a small fragment of single-stranded DNA or RNA (approximately 20 to 500 bp) that is used to detect a nucleic acid sequence complementary to it. Double-stranded DNA is heat denatured to become single-stranded and subsequently labeled with a radioisotope (usually phosphorus-32), a fluorochrome, or an enzyme (e.g., horseradish peroxidase) to become a probe.

Operation method

basic program

Materials and Instruments

Genes
Transcription buffer DTT RNAase inhibitor CTP ATP GTP S-UTP Ammonium acetate Ammonium acetate Ethanol
Water Bath Centrifuge Incubator Oven

Move

1. The target gene is inserted in a vector containing the SP6, T3 or T7 promoter. Enzymatic cleavage downstream of the coding sequence makes the plasmid linear. the DNA is extracted with phenol/chloroform, precipitated with ethanol, washed with 70% ethanol and air-dried. The precipitate was reconstituted in sterile water to 1 μg/μl.2. Prepare the following reaction mixture at room temperature (total volume 20 μl):
(1) 4.0 μl 5× transcription buffer

(2) 0.2 μl 1 mol/l DTT

(3) 60 U RNAase inhibitor

(4) 1.0 μl each of three 10 mmol/l NTPs

(5) 1.0 μl 1 μg/μl digested DNA

(6) 10.0 μl ^［ 35S］UTP

(7) 16 U SP6 or T7 RNA polymerase

(8) incubate at 37 ℃ for 30 min, add 16 U polymerase and incubate at 37 ℃ for 40 min.
3. Add 60 U RNAase inhibitor, 20 μl of 10 mg/ml carrier RNA and 1.0 μl of Enzyme 1 to the reaction mixture and incubate at 37°C for 10 min to remove the touch plate.
4. Add 0.8 μl of 1 mol/l DTT, 63 μl of sterile water and 10 μl of 3 mol/l sodium acetate to the reaction mixture and remove 1 μl to determine the cpm/μl value.

5. Add 36.4 μl of 7.5 mol/l ammonium acetate to the reaction mixture (final concentration 2 mol/l) plus 50~100 μg of tRNA carrier and 272 μl of cold anhydrous ethanol, precipitate on dry ice for 10 min, wash the precipitate with cold 70% ethanol and air dry, repeat this step if necessary. Resolve the DNA precipitate with 100 μl of 10 mmol/l DTT.

6. Determine the cpm/μl value and calculate the percentage of incorporation; the RNA probe should be used within 2 to 3 days. (70% to 90% labeled admixture results in 70 to 90 ng of labeled RNA).

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