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Important
If you have already prepared your cDNA, use 70-200 ng cDNA and start from the End-prep step.
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Prepare the RNA in nuclease-free water
- Transfer 100 ng PolyA+ RNA into a 1.5 ml Eppendorf DNA LoBind 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 a 0.2 ml PCR tube:
Reagent |
Volume |
poly A+ RNA, 100 ng |
x μl |
VNP |
2.5 μl |
10 mM dNTPs |
1 μl |
RNase-free water |
7.5-x 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 (SSP) |
2 μl |
Total |
8 μl |
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Mix gently by flicking the tube, and spin down.
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Add the strand-switching buffer to the snap-cooled, annealed mRNA, 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 |
∞ |
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