-
Prepare the RNA in nuclease-free water
- Transfer 1 ng PolyA+ RNA (or ~50 ng total RNA) into a 1.5 ml Eppendorf DNA LoBind tube
- Adjust the volume to up to 9 μl with nuclease-free water
- Mix by flicking the tube to avoid unwanted shearing
- Spin down briefly in a microfuge
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Prepare the following reaction in a 0.2 ml PCR tube:
Reagent |
Volume |
1 ng PolyA+ RNA (or ~50 ng total RNA) |
x μl |
VN Primers (VNP), at 2 µM |
1 μl |
10 mM dNTPs |
1 μl |
RNase-free water |
9-x μl |
Total volume |
11 μl |
-
Mix gently by flicking the tube, and spin down.
-
Incubate at 65°C for 5 minutes and then snap cool on a pre-chilled freezer block for 1 minute.
-
In a separate tube, mix together the following:
Reagent |
Volume |
5x RT Buffer |
4 μl |
RNaseOUT |
1 μl |
Nuclease-free water |
1 μl |
Strand-Switching Primer (SSP, at 10 µM) |
2 μl |
Total |
8 μl |
-
Mix gently by flicking the tube, and spin down.
-
Add the strand-switching buffer to the snap-cooled, annealed mRNA, mix by flicking the tube and spin down.
-
Incubate at 42°C for 2 minutes in the thermal cycler.
-
Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 µl.
-
Mix gently by flicking the tube, and spin down.
-
Incubate using the following protocol using a thermal cycler:
Cycle step |
Temperature |
Time |
No. of cycles |
Reverse transcription and strand-switching |
42°C |
90 mins |
1 |
Heat inactivation |
85°C |
5 mins |
1 |
Hold |
4°C |
∞ |
|