RNA labeling

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  1. Retrieve 50ug total RNA sample in 75% EtOH (-80C)
  2. Add 1/40 volume 3M NaOAc
  3. -80C 20-30 minutes
  4. Spin 14K rpm 10 minutes
  5. 100uL 70% EtOH
  6. Remove EtOH and air dry pellet
  7. Add 12.9uL dH2O (DEPC treated or RNase free)
  8. Add 2.5uL anchored oligo dT17 or random primer (2ug/uL)
  9. Heat the mixture to 70C 10 minutes
  10. Snap chill on ice
  11. Add 3uL Cy3 or Cy5 dCTP
  12. Add 11.6uL superscript mix
  13. 42C for 2 hours
  14. 1uL RNase A (10mg/mL)
  15. 1hr at 37C
  16. Qiagen PCR purification
  17. OD labeled DNA using Nanodrop

superscript mix 

Components	         1X	 5X
5X first strand buffer	 6uL     30uL
0.1M DTT                3uL     15uL
Superscript II          2uL     10uL
25mM dNTP mix           0.6uL   3uL
total volume            11.6uL  58uL
Retrieved from "http://genome.med.yale.edu/index.php/RNA_labeling"
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