First strand cDNA synthesis with Ready-To-Go Beads (Amersham)

Contributed by Dr.A.Gratchev

This cDNA synthesis system simplifies your work dramatically. All reaction components are premixed and lyophylised. You have to add your RNA and (for Your-Prime beads) the primer. Another advantage of the system is a little number of pipetting steps required, and therefore reduced risk of RNase contamination and RNA degradation. Company control of the product provides you with standard reaction conditions. The kit can be stored at RT. The only disadvantage which I observed is a bit lower reaction efficiency in comparison to the conventional reaction when you have to add all your components separately.

  1. Prepare you RNA (up to 5 ug) in the volume up to 25 ul.
  2. Denature RNA by incubating at 65 C for 10 min, transfer on ice.
  3. Optional (for Your-Prime beads). Add 10-100 pmol selected primer.
  4. Adjust volume to 33 ul.
  5. Transfer obtained volume to the tube with the beads, incubate for 1 min at RT.
  6. Vortex carefully and incubate for 1 h at 37 C.
  7. Inactivate reaction by heating to 65 C for 10 min, or freeze the reaction.
  8. Use nondiluted as well as 1:10 and 1:100 dilutions for PCR.

You can control your cDNA preparaion for reaction efficiency and genomic DNA contamination using protocol provided here.

\METHODS\cDNA\Ready to Go Beads (Amersham)