Biology Reference
In-Depth Information
Protocol 1 GENERATION AND LABELLING OF STRAND-SPECIFIC CDNA
FROM PROKARYOTIC TOTAL RNA—Cont'd
6. Open the cap of the 2-ml receptacle tube, remove and retain the microspin cup,
and discard the DNA-binding solution containing the uncoupled dye.
7. Combine 100 ml of the DNA-binding solution and 100 ml of 70% (v/v) ethanol. Mix
well by vortexing. Make sure that the two solutions are well mixed prior to use.
8. Add the 200 ml of DNA-binding solution/ethanol mixture to the microspin cup.
Snap the cap of the receptacle tube onto the top of the microspin cup.
9. Centrifuge the tube in a microcentrifuge at maximum speed for 30 s.
10. Open the cap of the 2-ml receptacle tube, remove and retain the microspin cup,
and discard the DNA-binding solution/ethanol mixture.
11. Add 750 ml of 75% ethanol to the microspin cup. Snap the cap of the receptacle
tube onto the top of the microspin cup.
12. Centrifuge the tube in a microcentrifuge at maximum speed for 30 s.
13. Open the cap of the 2-ml receptacle tube, remove and retain the microspin cup,
and discard the wash buffer.
14. Repeat steps 11-13.
15. Place the microspin cup back in the 2-ml receptacle tube and snap the cap onto
the microspin cup.
16. Centrifuge the tube in a microcentrifuge at maximum speed for 1 min.
17. Transfer the microspin cup to a fresh 1.5-ml microcentrifuge tube and discard the
2-ml receptacle tube.
18. Add 50 ml of 10 mM Tris base, pH 8.5 directly onto the top of the fibre matrix at
the bottom of the microspin cup.
19. Incubate the tube at room temperature, in the dark, for 5 min.
20. Snap the cap of the 1.5-ml microcentrifuge tube onto the microspin cup and
centrifuge the tube in a microcentrifuge at maximum speed for 30 s.
21. Open the lid of the microcentrifuge tube and recover the flow through containing
the purified labelled cDNA.
22. Elute additional labelled cDNA by pipetting the flow through back onto the fibre
matrix of the same microspin cup.
23. Re-seat the spin cup on the same 2-ml receptacle tube that contained the liquid
from the first-pass elution.
24. Incubate the tube at room temperature for 5 min.
25. Snap the cap of the 1.5-ml microcentrifuge tube onto the microspin cup and
centrifuge the tube in a microcentrifuge at maximum speed for 30 s.
26. Open the lid of the microcentrifuge tube and recover the flow through containing
the purified labelled cDNA.
27. Harvest one final elution from the microspin cup by repeating steps 22-26.
28. Open the lid of the microcentrifuge tube and recover the flow through containing
the purified labelled cDNA.
Step 5. Spectrophotometric Quantitation and Quality Control of cDNA
1. Determine cDNA and Cy3 dye concentrations by using a small-volume
spectrophotometer (such as a NanoDrop instrument).
2. Quantitate each cDNA sample according to the following formula:
cDNA concentration (ng/ml)¼A26033Dilution Factor
3. Verify that all samples meet the following requirements:
Concentration 20 ng/ml
A260/A2801.8
A260/A2301.8
