Reverse Transcription with Maxima (cDNA)
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Before starting: Warm dry bath 1 to 65C and dry bath 2 to 50C.
Clean work space with ethanol.
Prepare bucket with ice.
Defrost samples and working solutions on ice.
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RNA must be kept on ice always.
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Work with clean/filtered pipettes only.
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Primer working solution: 77µl HPLC DDW and 3 µl primer.
1. Prepare mix 1 :
Mix 1
6.5 µl HPLC DDW
1 µl DNTP’s (40mM)
1.5 µl reverse primer
Total: 15 µl (including RNA).
2. Prepare 1.5 tube with 9 µl mix 1.
3. Add 6 µl RNA, spindown.
4. Incubate at 65C for 5 min.
5. Prepare mix 2:
Mix 2
Add in following order:
4 µl Maxima Buffer X5
0.5 µl RNase inhibitor
0.2 - 0.4 µl Maxima enzyme
Total: 20 µl (including mix 1 and RNA).
6. Remove RT tube from 65C , and incubate on ice 2 min, spindown.
7. Warm dry bath 1 to 85C.
8. On ice, add 5 µl of mix 2 to each tube, spindown.
9. Incubate RT tube at 50C for 30min.
10. Incubate RT tube at 85C for 5 min.
11. Store at -20C, avoid frequent thaw-freeze cycles.
Reverse Transcription with methyl mercury
1. Prepare 1.5 ml tube with 4.5 µl RNA
2. In hood, add 0.5 µl methyl mercury (0.1 M), mix gently and spin.
3. Incubate 10 min at room temp.
4. In hood, add 0.5 µl β-mercaptoethanol (1.4M).
5. On ice add regents as above, and omit 65C step.
Reverse Transcription with random primers
Prepare random primer (500 µg/ml) – 7.5 µl primer + 7.5 µl DDW HPLC
Use 0.5 µl random primer for mix 1 (instead of 1.5 µl reverse primer).
Incubate at 65C, 5 min.
Add mix 2.
Incubate: 25C, 2 min
50C, 30 min
85C, 5 min