Synthesis of cDNA probes from mRNA using random oligonucleotide primers
In this protocol, poly(A)+ RNA is used as a template to synthesize cDNA probes by random priming reaction, and these probes can be used for differential screening of cDNA libraries. This experiment is based on the "Guide to Molecular Cloning Experiments, Third Edition", translated by Huang Peitang et al.
Operation method
Synthesis of cDNA probes from mRNA using random oligonucleotide primers
Principle
In this protocol, poly(A)+RNA is used as a template to synthesize cDNA probes by random priming reactions, and these probes can be used for differential screening of cDNA libraries.
Materials and Instruments
Reverse transcriptase Reverse transcriptase buffer Random deoxyribonucleotide primer Template mRNA Move I. Materials For more product details, please visit Aladdin Scientific website.
Ammonium acetate DTT EDTA Ethanol HCl NaOH Phenol Chloroform Tryptic RNAase inhibitor SDS Tris-Cl dNTP solution
Ice-water bath Sephadex G-50 centrifuge column Water bath or heated 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)
NaOH ( 3 mol/L)
Phenol: chloroform (1:1, V/V)
Tryptic RNAase inhibitor (20 units/μl )
SDS ( 10% m/V)
Tris-Cl ( 1 mol/L, pH 7.4)
2. Enzyme and buffer
reverse transcriptase
10X reverse transcriptase buffer
3. Nucleic acids and oligonucleotides
dNTP solution containing 20 mmol/L each of dATP, dGTP and dTTP.
dCTP (125 μmol/L)
Random deoxynucleotide primers of six or seven bases in length.
Template mRNA
4. radioactive compounds
[ α-32P ] dCTP ( 10 mCi/ml, specific activity >3000 Ci/mmol)
5. Specialized equipment
Ice water bath
Sephadex G-50 centrifuge column, equilibrated with TE (pH 7.6)
Preheated water bath or heating plate to 45°C, 68°C and 70°C
II. Methods
1. Transfer 1 μg poly(A )+RNA to a sterilized microcentrifuge tube. Bring the volume of solution to 4 μl with RNAase-free water, cap the microcentrifuge tube tightly, heat at 70 °C for 5 min, and then quickly transfer the tube to an ice water bath.
2. Add to the cold solution in the microcentrifuge tube:
10 mmol/L DTT 2.5 μl
Placental RNAase Inhibitor 20 units
Random deoxy oligonucleotide primer 5 μl
10X reverse transcription buffer 2.5 μl
20 mmol/L dGTP, dATP and dTTP solution 1 μl
125 μmol/L dCTP solution 1 μl
10 mCi/ml [ α-32P ] dCTP
(specific activity > 3000 Ci/mmol) 10 μl
Water without RNAase to 24 μl
Reverse transcriptase (200 units) 1 μl
3. Add the following reagents to terminate the reaction:
0.5 mol/L EDTA ( pH 8.0) 1 μl
10% (m/V) SDS 1 μl
Mix the reagents in the tube thoroughly.
4. Add 3 μl of 3 mol/L NaOH to the reaction tube and incubate the mixture at 68°C for 30 min to hydrolyze the RNA.
5. Cool the reaction mixture to room temperature, add 10 μl of 1 mol/L Tris-Cl (pH 7.4) to neutralize the solution, mix well, and then add 3 μl of 2.5 mol/L HCl. Take a very small drop of the liquid on pH paper to check the pH of the solution.
6. Purify the cDNA by phenol:chloroform extraction.
7. Selectively precipitate the radiolabeled probe by centrifugal column chromatography or ethanol in the presence of 2.5 moI/L ammonium acetate to separate it from the unadulterated dNTP.
8. Determine the percentage of radiolabeled dNTP incorporation by trichloroacetic acid (TCA) precipitation or DE-81 membrane binding.