Reverse transcription

RNA. 2µL (500ng/µl). 2µl (250ng/µl). RH‡‡ (OdT§§). 2µl (1µl). 1µl (0.5µl). 10mM dNTPs. 1µl. 0.5µl. H20. 7.75µl (8.75µl). 2.875µl (3.475µl). Denaturate at 65°C ...
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Analysis of nucleic acids

Reverse transcription Do not forget Ctrl without RNA and another with RNA but without RT.

1- Superscript II (Invitrogen)

In each tube add: 20µl

10µl

2µL (500ng/µl)

2µl (250ng/µl)

RH (OdT )

2µl (1µl)

1µl (0.5µl)

10mM dNTPs

1µl

0.5µl

7.75µl (8.75µl)

2.875µl (3.475µl)

RNA ‡‡

§§

H20

Denaturate at 65°C for 10 minutes and quick-chill on ice. Centrifuge briefly.

Add to each tube: 20µl

10µl

5X buffer

4µl

2µl

0.1M DTT

2µl

1µl

1µl

0.5µl

0.25µl

0.125µl

RNAseout*** Reverse transcriptase

†††

Incubate at 25°C for 10 minutes | Incubate at 42°C for 50 minutes |

in a thermal cycler with heated lid @ 70°C

Incubate at 70°C for 15 minutes |

Store RT at –20°C

‡‡

50ng/µl (Invitrogen) Anchored oligo-dT 500µg/ml *** Invitrogen ††† 200 U/µL §§

Analysis of Nucleic Acids 12