DNA cleaning RT-PCR

WAV, 07/10/10

DNA Cleaning DNAFree from Ambion

RNA                                 5 µl (10-20 µg)

10 X Buffer                      2 µl

DNAse                              1µl

RNAse free Water            12 µl

1. Incubate for 30 min at 37^o C
2. Add 5 µl of DNAse inhibiting buffer and incubate for 2 min at room temperature (flip the tube two or three times).
3. Centrifuge for 1 min at 13000 rpm (Top bench centrifuge)
4. Transfer supernatant to a fresh tube.
5. Quantify on nanodrop.

Reverse Transcription Reaction (Using M-MLV-RT and RNAsin from promega)

Mix:

Total RNA                              2.5-5 µg

10 mM oligodT                    2 µl

DEPC water up to                  10 µl

Incubate 5-10 min at 70 ^o C

Transfer for 5 min to ice.

Add: 5 x RT Buffer                   5 µl

2.5 mM dNTP                   5 µl

40 U/ µl RNAsin                   0.5 µl

200U/ µl M-MLV-RT               1 µl

Water                         3.5 µl

Spin and then incubate for 1 h at 47^o C.