Random priming method (radiolabeling of DNA using random oligonucleotide extensions in the presence of melted agarose)

Summary

This method can be used for radiolabeling of DNA recovered from low melting point agarose gels (Feinberg and Vogelstein 1983, 1984). This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.

Operation method

Random priming method (radiolabeling of DNA using random oligonucleotide extensions in the presence of melted agarose)

Principle

This method can be used for the recovery of radiolabeled DNA from low melting point agarose gels (Feinberg and Vogelstein 1983, 1984).

Materials and Instruments

E. coli DNA Polymerase I Klenow Fragment Template DNA
Ammonia acetate Bovine serum albumin Ethanol Ethidium bromide NA Termination Storage buffer Random primer buffer
Basic agarose gel or denaturing polyacrylamide gel Boiling water bath Sephadex G-50 centrifuge columns

Move

I. Materials

1. Buffers and solutions

Ammonia acetate (10 mol/L)

Bovine serum albumin (10 mg/ml)

Ethanol

Ethidium bromide (10 mg/ml) or SYBR Gold dye

NA termination/storage buffer (50 mmol/L Tris-Cl (pH 7.5), 50 mmol/L NaCl, 5 mmol/L EDTA (pH 8.0), 0.5% (m/V) SDS)

5X random primer buffer (250 mmol/L Tris (pH 8.0), 25 mmol/L MgCl₂, 20 mmol/L DTT, 2 mmol/L each of unlabeled dNTP, 1 mol/L HEPES (adjusted to pH 6.6 with 4 mol/L NaOH), 1 mg/ml random deoxy-oligonucleotide primer of 6 bases in length )

2. Enzyme and buffer

E. coli DNA polymerase Ⅰ Klenow fragment

3. gel

Basic agarose gel or denaturing polyacrylamide gel

4. Nucleic acids and oligonucleotides

Random deoxynucleotide primers of 6 or 7 bases in length (125 ng/μl TE, pH 7.6 )

Template DNA

5. Radiocomplexes

[ ^α-32P ] dNTP ( 10 mCi/ml, specific activity >3000 Ci/mmol)

6. Specialized equipment

Boiling water bath

Sephadex G-50 centrifuge column equilibrated with TE ( pH 7.6)

II. Methods

1. After electrophoresis, stain the gel with ethidium bromide (final concentration 0.5 μg/ml) or SYBR Gold and cut off the desired bands, removing as much excess gel as possible.

2. Place the cut bands into pre-weighed microcentrifuge tubes and weigh the bands. Add 3 ml of water per gram of agarose gel.

3. Place the microcentrifuge tube in a boiling water bath for 7 min to melt the gel and denature the DNA.

If labeling is to be done immediately, the tubes can be placed at 37°C until template addition is required; otherwise, they can be stored at -20°C. However, each time the tubes are removed from -20°C, they can be stored at -20°C for a few minutes. However, after each removal from -20°C, the gel mass containing DNA should be heated at 100°C for 3-5 min and then placed at 37°C until the radiolabeling reaction begins.

4. Place a new microcentrifuge tube in a 37°C water bath or heating plate and add in the following order:

5X Oligonucleotide Labeling Buffer 10 μl

10 mg/ml bovine serum albumin solution 2 μl

DNA (volume not exceeding 32 μl) 20~50 ng

10 mCi/ml [ ^α-32P ] dNTP 5 μl

( Specific activity >3000 Ci/mmol)

Klenow fragment (5 units) 1 μl

Water Add to 50 μl

Mix thoroughly with a microsampler and incubate at room temperature for 2-3 h or at 37℃ for 60 min.

5. Add 10 μl of NA termination/storage buffer to the reaction solution.

Store the radiolabeled probe at -20°C for hybridization.

OR

Separate radiolabeled probe from unbound dNTP by centrifugal column chromatography or selectively precipitate radiolabeled DNA with ammonium acetate and ethanol; this step may be omitted if >50% of the radiolabeled dNTP has been incorporated into the reaction.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/