Reverse Transcriptase (RT) Master Mix

RT 2X Master Mix

• Up to 9kb cDNA synthesis
• Ensures sample to sample consistency
• Large RNA sample volume capacity
• Ready to use

Size: 100 rxns

RT 2X Master Mix is a proprietary, ready-to-use master mix for first-strand cDNA synthesis in a 2X
concentration. This optimized reaction mix contains ribonuclease inhibitor, dNTPs, and a balanced
concentration for oligo(dT) and random primers. The ribonuclease inhibitor effectively protects RNA
template from degradation. The oligo(dT) anneals selectively to the poly(A) tail of mRNAs and the
random primers do not require the presence of poly(A) and they are utilized for the transcription of
mRNA 5-end regions. The resultant cDNA can be directly used as template in different PCR experiments.

Kit Components:

Storage: 
Store at -20°C in a frost-free freezer.

• Application:

  • cDNA synthesis
  • Construction of cDNA libraries
  • Generation of probes for hybridization

   

  Protocol:

  1. Thaw RNA templates and all reagents on ice. Mix each solution by vortexing.
  2. Assemble the following components in a tube on ice, and mix well:

  Components	Volume	Final Conc.
  Total RNA, or	Variable	1 ng - 2 µg/rxn
  mRNA	Variable	1 pg - 2 ng/rxn
  2X Reaction Mix	10 µl	1X
  H2O	Up to 19 µl	-

  3. Heat the mixture at 65°C for 5 mins and incubate on ice for at least 1 min.
  4. Collect all components by a brief centrifugation and add 1 µl of the EasyScript RTase to the tube.
  5. Mix well and collect all the components by a brief centrifugation.
  6. Incubate the tube at room temperature for 10 min for annealing.
  7. Perform cDNA synthesis by incubating the tube for 50 min at 42°C.
  8. Stop the reaction by heating it at 85°C for 5 min.
  9. Chill on ice. The newly synthesized first-strand cDNA is ready for immediate downstream applications.