CDNA Synthesis and Labeling

1. Prepare two labeled cDNAs, a test and a reference, with FITC and Biotin, respectively.

2. Transfer one |g of total RNA in 13 |L DEPC-treated water to each tube.

3. Add 5 |L of a 10-fold-diluted control RNA solution (Micromax TSA kit) into the tube.

4. Add 1 ||L each of Reaction Mix Concentrate (Micromax TSA kit) and FITC- or Biotinlabeled nucleotide.

5. Incubate the tubes at 65°C for 10 min, then cool the reaction mixture to room temperature for 5 min.

7. Add 2.5 |L 10X RT Reaction Buffer (Micromax TSA kit), then mix well.

8. Add 2 |L AMV-RT/RNase Inhibitor Mix (Micromax TSA kit).

10. Cool the tube to 4°C for 5 min, add 2.5 |L each of 0.5 M EDTA, pH 8.0, and 1 N NaOH, then mix well.

11. Incubate 65°C for 30 min (do not exceed), then cool the tube to 4°C.

12. Add 2.7 |L 5 M NH4Ac and 31 |L isopropanol, vortex and incubate at 4°C for 30 min.

13. Centrifuge for 20 min at 10,000g at 4°C, and carefully decant the supernatant.

14. Wash the pellet twice with 70% ethanol, and dry in a vacuum concentrator.

15. Resuspend the pellet in 20 |L Hybridization Buffer (Micromax TSA kit). Store at -20°C.

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