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Important
If you have already prepared your cDNA, use 70–200 fmol cDNA (~70–200 ng if your sample is 1.5 kb) and start from the cDNA repair and end-prep step.
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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 |
User-supplied VN Primer diluted to 2 µM |
✓ |
✓ |
✓ |
User-supplied Strand-Switching Primer diluted to 10 µM |
✓ |
✓ |
✓ |
10 mM dNTP solution |
✓ |
✓ |
✓ |
RNaseOUT |
Not frozen |
✓ |
✓ |
Maxima H Minus Reverse Transcriptase |
Not frozen |
✓ |
✓ |
Maxima H Minus 5x RT Buffer |
✓ |
✓ |
Mix by vortexing |
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Prepare the RNA in nuclease-free water
- Transfer 100 ng Poly(A)+ RNA or 1 μg of total RNA into a 0.2 ml PCR tube
- Adjust the volume to up to 7.5 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 the 0.2 ml PCR tube containing the prepared RNA input:
Reagent |
Volume |
RNA input (100 ng Poly(A)+ RNA or 1 μg of total RNA) from step above |
7.5 μl |
VN Primer diluted to 2 μM |
2.5 μl |
10 mM dNTPs |
1 μl |
Total volume |
11 μl |
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Mix gently by flicking the tube, and spin down.
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Incubate at 65°C for 5 minutes and then snap cool on a pre-chilled freezer block for 1 minute.
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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 diluted to 10 µM |
2 μl |
Total |
8 μl |
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Mix gently by flicking the tube, and spin down.
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Add the 8 μl of strand-switching reagents (prepared in steps 6-7) to the 11 μl of snap-cooled mRNA (from steps 2-5). Mix by flicking the tube and spin down.
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Incubate at 42°C for 2 minutes in the thermal cycler.
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Add 1 µl of Maxima H Minus Reverse Transcriptase. The total volume is now 20 µl.
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Mix gently by flicking the tube, and spin down.
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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 |
∞ |
|