Protocol for TotalScript™ RNA-Seq Kit

TotalScript RNA-Seq Kit
7. Appendix
Appendix A. Bioanalyzer Profile Of A TotalScript RNA-Seq Library
A TotalScript RNA-Seq library was made from 5 ng of random primed Universal Human
Reference RNA (UHR) using the TotalScript Optimized Buffer as described in Part 3.A of
the procedure.
Appendix B. It is recommended to use the Control RNA as a positive control for library
prep to determine that all the steps of the protocol are followed as directed.
1. Thaw the Control RNA and keep on ice. Remove 1 ul of the Control RNA and place
in a fresh RNase-free tube, then add 99 ul of Nuclease-free water and mix well. Keep
diluted Control RNA on ice.
2. Set up the following reaction, as per step 3.A.1. of the TotalScript cDNA Synthesis
procedure:
2 μl 1:100 diluted Control RNA
2.5 μl TotalScript First-Strand Buffer
1 μl Random Hexamer Primers
12.5 μl Nuclease-free water
18.0 μl Total
3. Place the samples in a thermocycler with a heated lid. Heat for 2 minutes at 65°C,
then hold at 4°C.
4. Proceed to Part 3.B, 1st Strand cDNA synthesis.
5. At Step 4.E. PCR Amplification:
a. Amplify the sample for 12 cycles and purify the reaction as directed in Part 4.F. of
the protocol.
b. Run 1 ul on a bioanalyzer using a high sensitivity chip.
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