-
Thaw the following reagents and spin down briefly using a microfuge, before mixing as indicated in the table below, and place on ice.
Reagent |
1. Thaw at room temperature |
2. Briefly spin down |
3. Mix well by pipetting |
Custom-ordered sequence-specific primer |
✓ |
✓ |
✓ |
Strand Switching Primer II (SSPII) |
✓ |
✓ |
✓ |
RNaseOUT |
Not frozen |
✓ |
✓ |
10 mM dNTP solution |
✓ |
✓ |
✓ |
Maxima H Minus Reverse Transcriptase |
Not frozen |
✓ |
✓ |
Maxima H Minus 5x RT Buffer |
✓ |
✓ |
Mix by vortexing |
-
Prepare the RNA in nuclease-free water.
- Transfer 4 ng Poly(A)+ RNA, or 200 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
-
Prepare the following reaction in a 0.2 ml PCR tube:
Reagent |
Volume |
RNA |
9 μl |
Custom-ordered sequence-specific primer, diluted to 2 μM |
1 μl |
10 mM dNTPs |
1 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.
-
To the same 0.2 ml PCR tube, add the following:
Reagent |
Volume |
Maxima H Minus 5x RT Buffer |
4 μl |
RNaseOUT |
1 μl |
Nuclease-free water |
1 μl |
Strand Switching Primer II (SSPII) |
2 μl |
Total (including all reagents) |
19 μl |
-
Tip
Strand Switching Primer II (SSPII) base pairs to the deoxycytidine present at the 5' end of the first cDNA strand synthesised. This allows the reverse transcriptase to "strand-switch" for synthesis of the second cDNA strand.
-
Mix gently 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 |
∞ |
|
-
End of step
Take your samples forward into the next step. However, at this point it is also possible to store the sample at -20°C overnight.