Reverse Transcription of RNA

From CCGB

HardisonProtocols

Reverse Transcription of RNA

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    -- by Hao Wang

1. Add TE to 4 μl aliquot of RNA to give final concentration of 50 ng/μl.

2. Make up a master mix (keep on ice).

                                       ×8
  5 × M-MLV RT buffer		        4 μl				32
  Random Hesas (0.1 μg/μl)		2 μl				16
  0.1 M DTT				2 μl				16
  M-MLV RT (200 u/μl)		        2 μl				16
  RNasin (20-40 u/μl)		        0.5 μl				8
  10mM dNTPs				0.4 μl				3.2
  dd RNase free H2O			1.1 μl 				8.8
  Total				12 μl				96

3. Label 0.5 ml microtubes before starting RT reaction and place on ice.

4. Aliquot 12 μl of master ix into each tube.

5. Aliquot 8 μl of RNA samples (@50ng/μl) into each tube and mix (v=20 μl, 400 ng RNA total). Spin short.

6. Incubate reaction @ 37 °C for 30 min. Spin short.

7. Place tubes on ice and add 80 μl TE. Mix by tapping.

8. Store @ –20 °C (this is stock solution).

9. For RT-PCR, try first one or two clones (RTs) using undiluted samples and 1:10 dilution to see which one gives better product.