Synthesis of radiolabeled minus cDNA probes using oligo (dT) as primer
This protocol describes the preparation of a deduction cDNA probe by hybridization with an mRNA driver followed by purification of single-stranded radiolabeled cDNA by hydroxyapatite chromatography. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Synthesis of radiolabeled minus cDNA probes using oligo (dT) as primer
Principle
This protocol describes the preparation of a minus cDNA probe by hybridization with an mRNA driver followed by hydroxyapatite chromatography purification of a single-stranded radiolabeled cDNA.
Materials and Instruments
Reverse transcriptase Reverse transcriptase buffer Driver mRNA Template mRNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate DTT EDTA Ethanol HCl Isobutanol NaOH Phenol Chloroform Placenta RNA enzyme inhibitor SDS SDS EDTA solution Phosphate buffer SPS buffer dNTP solution
Boiling water bath Ice water bath Mineral oil Sephadex G-50 column Microcentrifuge tubes Water bath or heating plate
1. Buffers and solutions
Ammonium acetate (10 mol/L)
DTT (1 mol/L)
EDTA ( 0.5 mol/L, pH 8.0)
Ethanol
HCl ( 2.5 mol/L)
Isobutanol
NaOH ( 3 mol/L)
Phenol: chloroform (1:1, V/V)
Placental RNAase inhibitor (20 units/μl)
SDS ( 10% m/V)
SDS/EDTA solution (30 mmol/L EDTA (pH 8.0), 1.2% SDS)
Phosphate buffer (2 mol/L, pH 6.8)
SPS buffer (0.12 mol/L (pH 6.8), 0.1% (m/V) SDS)
Tris-Cl ( 1 mol/L, pH 7.4)
2. Enzyme and buffer
reverse transcriptase
10X reverse transcriptase buffer
3. Nucleic acids and oligonucleotides
dCTP ( 125 μmol/L)
dNTP solution containing 20 mmol/L each of dATP, dGTP, and dTTP.
Driver mRNA
oligo (dT )12-18
Template mRNA
4. radioactive compounds
[ α-32P ] dCTP ( 10 mCi/ml, specific activity >3000 Ci/mmol)
5. Specialized equipment
Hydroxyapatite chromatography device for separation of single and double stranded nucleic acids at 60 °C.
Boiling water bath
Ice water bath
Mineral oil, pipette tips with elongated tips that can be attached to the autosampler, or siliconized disposable 20 μl glass capillaries, files or diamond knives
Sephadex G-50 column (5 ml) equilibrated with TE (pH 8.0)
Sephadex G-50 column, equilibrated with TE (pH 8.0) containing 0.1% SDS
Siliconized microcentrifuge tubes (1.5 ml)
Preheated water bath or heating plate to 45°C, 68°C and 70°C
II. Methods
1. Transfer 5-10 μg of poIy(A )+ RNA into a sterilized microcentrifuge tube and bring the volume of poIy(A )+ RNA to 40 μl with RNAase-free water. cap the tube tightly and heat at 70°C for 5 min, then quickly transfer the microtube to an ice-water bath.
2. Add to the cooled microtube:
0.1 mol/L DTT 2.5 μl.
Placental RNAase Inhibitor 200 units
oligo (dT )12-18 10 μl
10X reverse transcription buffer 25 μl
20 mmol/L dGTP, dATP, dTTP solution 10 μl
125 μmol/L dCTP 10 μl
10 mCi/ml [ α-32P ] dCTP
(specific activity > 3000 Ci/mmol) 100 μl
Water without RNAase to 240 μl
Reverse transcriptase (2000 units) 10 μl
Tap the outside of the tube to mix the components, centrifuge slightly in a microcentrifuge to collect the reaction mixture at the bottom of the tube, and incubate the reaction for 1 h at 45°C.
3. Add the following reagents to terminate the reaction:
0.5 mol/L EDTA (pH 8.0) 10 μl
10% (m/V) SDS 10 μl
'Mix the reagents in the tube.
4. Add 30 μl of 3 mol/L NaOH to the reaction tube and incubate the reaction mixture at 68°C for 30 min to hydrolyze the RNA.
5. Cool the reaction mixture to room temperature, add 100 μl of 1 mol/L Tris-Cl (pH 7.4) neutralizing solution, mix well, and then add 30 μl of 2.5 mol/L HCl. Spot a small drop (< 1 μl) of the solution on pH paper to check its pH.
6. Purify the cDNA by phenol:chloroform extraction.
7. Detect the proportion of radiolabeled dNTP incorporated by TCA precipitation or DE-81 membrane binding. Calculate the yield of cDNA according to the following formula:
In a reaction containing 1.5 nmol of limited dNTP: 
8. Separate the radiolabeled probe from the unadopted dNTP by chromatography on a 5 ml Sephadex G-50 column.
9. To the radiolabeled cDNA, add 10 times the weight of the driver RNA, minus the cDNA probe, and add 0.2 volume of 10 mol/L ammonium acetate and 2.5 volume of ice-cold ethanol. Allow to stand at 0°C for 10-15 min, then centrifuge in a microcentrifuge for 5 min at 4°C at maximum speed to recover nucleic acids.
10. Pipette off all the ethanol, uncap the tube and place it on a test bench to evaporate the remaining ethanol. Dissolve the nucleic acid in 6 μl of RNAase-free water.
11. Add to the dissolved nucleic acid:
2 mol/L sodium phosphate (pH 6.8) 2 μl
SDS/EDTA solution 2 μl
12. Cover the solution with a drop of light mineral oil or draw the mixture into a siliconized disposable 20 μl glass capillary and seal the ends of the capillary over a Bunsen burner flame.
13. Place the microcentrifuge tube or sealed capillary in a boiling water bath for 5 min and transfer to a 68°C water bath for hybridization up to Cr0t = 1000 mol-s/L. 
14. Remove the microcentrifuge tube or capillary from the water bath. Using a pipette tip with an elongated tip attached to a microsampler, aspirate the hybridization solution from the microcentrifuge tube or open the end of the capillary with a file or diamond pen and transfer the hybridization reaction solution to a tube containing 1 ml of SPS buffer.
15. Separate single-stranded and double-stranded nucleic acids by hydroxyapatite chromatography at 60 °C.
16. Combine the single-stranded cDNA-containing fractions and concentrate by repeated extraction with isobutanol: add an equal amount of isobutanol, mix the two phases by vortexing and shaking, and then centrifuge for 2 min in a microcentrifuge at maximal speed at room temperature, discarding the upper (organic) phase. Extraction with isobutanol was repeated until the volume of the aqueous phase was <100 μl.
17. Salt was removed from the cDNA by chromatography on a Sephadex G-50 centrifugal column equilibrated with TE (pH 8.0) containing 0.1% SDS.
18. Detect the amount of radioactivity in the sample and calculate the weight of the DNA minus the probe.
19. Repeat steps 9 to 18.
10% to 30% of the cDNA will form hybrids with the driver RNA in the second round of hybridization.