Molecular Techniques and Methods

Reverse Transcription From mRNA

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

This method generates high specific activity single stranded-cDNA probe from RNA template for use in subsequent applications (such as, differential screening of cDNA library).




MATERIALS AND SOLUTIONS

10 x Reverse Transcriptase Buffer (1 ml)

100 mM Tris-HCl (pH 8.8) --------------------- 100 ul of 1 M Tris-HCl
500 mM KCl ----------------------------------- 500 ul of 1 M KCl
1% Triton X-100 ------------------------------- 20 ul of 50% Triton X-100
Deionized H2O --------------------------------- 380 ul


RNA Hydrolysis Buffer (1 ml)

400 mM NaOH ------------------------------- 80 ul of 5 M NaOH
40 mM EDTA --------------------------------- 2 ul of 0.5 M EDTA
Deionized H2O -------------------------------- 918 ul


Reverse Transcriptase Stop Buffer (1 ml)

20 mM Tris-HCl (pH8.0) -------------------- 20 ul of 1 M Tris-HCl
2% SDS ------------------------------------- 200 ul of 10% SDS
50 mM EDTA ------------------------------- 100 ul of 0.5 M EDTA
200 mM NaCl ------------------------------- 40 ul of 5 M NaCl
Deionized H2O ------------------------------ 640 ul.




PROCEDURES

1. Add the following in a RNase-free microfuge tube.

10 x Reverse Transcriptase Buffer
2 ul
0.1 M DTT
2 ul
10 mM dNTPs w/o dCTP
1 ul
0.5 ug/ul Oligo dT18
1 ul
RNase Inhibitor (1 u/ul)
0.5 ul
Poly A+-mRNA (Total RNA)
0.1-0.5 ug (1-10 ug)
[a-32P]dCTP (800 Ci/mmole and 10 mCi/ml)
10 ul
Add DEPC-treated H2O to make a final volume of
20 ul


2. Denature the reaction mixture at 65oC for 5 min.

3. Add 1 ul of Superscript Reverse Transcriptase.

4. Incubate at 37-42oC for 1 hr.

5. Add 20 ul of RNA Hydrolysis Buffer.

6. Incubate at 70oC for 10 min.

7. Add 20 ul of 2 M Tris-HCl (pH7.0) and 40 ul of Reverse Transcriptase Stop Buffer.

8. Remove unincorporated [a-32P]dATP.

9. Continue to a hybridization experiment.




NOTES

  • In stead of oligo dT(12-18) priming, random primer can be used for cDNA synthesis.
  • Add 0.5 ul of 9 U A260 (5 ug/ul) pd(N)6 in the cDNA synthesis reaction.



  • KIT INFORMATION



    REFERENCES


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