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5'-RACE (Rapid Amplification of mRNA ends by PCR) clones 5'-ends of mRNA.
Database search can sometimes be used to find mRNA ends. See
EST assembler for more information.
Synthesis of first strand cDNA
1. First strand cDNA is synthesized using a gene-specific primer (reverse).
2. Heat RNA (0.1-5 mg mRNA or 5-20 mg total RNA)
to 65 °C.
3. Set up a reverse transcription reaction.
| MgCl2, 25mM | 4 µl |
Reverse Transcription 10X Buffer
(1X = 10 mM Tris-HCl pH 8.8, 50 mM KCl, and 0.1% Triton® X-100) | 2 µl |
| 10 mM dNTP mixture | 2 µl |
| rRNasin® Ribonuclease Inhibitor | 0.5 µl |
| AMV Reverse Transcriptase (H.C.) (15 units) | 1.5 µl |
| Gene-specific primer (0.5 µg) | 1 µl |
| RNA (0.5-20 µg) | 9 µl |
| Total volume | 20 µl |
4. Incubate the reaction at 42 °C for 1 hour.
5. Heat inactivate at 95 °C for 5 minutes.
Remove primers
6. Remove gene specific primers using one of the following methods:
PCR cleanup --- Microcon
PCR cleanup --- Qiaquick spin column
Purification of DNA from PAGE gels
Tailing
7. Set up a termianl transferase reaction. Tail with dGTP (or dATP).
| first strand cDNA |
12 ml |
| 100 mM dGTP (or dATP) |
2 ml |
5X Reaction Buffer
(1 M potassium cacodylate, 125 mM Tris-HCl pH 7.2,
0.05% Triton X-100 and 5 mM CoCl2) |
4 ml |
| Terminal Deoxynucleotidyl Transferase (20 units/ml) |
2 ml |
| Total volume |
20 ml |
8. Incubate at 37 °C for 10-30 minutes.
9. Heat inactivate at 65 °C for 10 minutes.
10. Dilute with TE.
11. Extract with phenol:chloroform:isoamyl alchol.
12. Add 1/10 volume of 3 M sodium acetate and precipitate with
2 volumes of ethanol.
13. Centrifuge and wash the pellet with 70% ethanol.
14. Air-dry and resuspend the pellet in 10-20 m
of TE or ddH2O.
PCR
15. PCR using 1-10 ml of tailed cDNA,
oligo (dC) (15-17mer) primer and gene-specific primer.
16. Run for 35-40 cycles on a thermocycler.
17. Run a second round PCR if necessary.
18. Analyze by agarose gel electrophoresis and sequencing.
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