clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual
D. Protocol: First-Strand cDNA Synthesis
The two 20 µl reactions described in the protocol convert 10 ng–1 µg of total or poly A+ RNA into
RACE-Ready first-strand cDNA
We recommend that you use poly A+ RNA whenever possible. However, if you have less than 50 µg of
total RNA we do not recommend purification of poly A+ RNA because the final yield will be too small to
effectively analyze the RNA quantity and quality.
We strongly recommend that you perform a positive control cDNA synthesis using the included
Mouse Heart Total RNA in addition to your experimental reactions. NOTE: If your RNA template is from a non-eukaryotic organism and/or lacks a polyadenylated tail,
follow the protocol for 5’-first-strand cDNA synthesis with random primers in Appendix D.
For 3’-first-strand cDNA synthesis, add a poly(A) tail using Poly(A) Polymerase (Takara Cat. No.
2180A), and proceed with the following protocol.
IMPORTANT:
Prior to cDNA synthesis, please make sure that your RNA is intact and free of contaminants (see
Section V.C. Assessing the Quality of the RNA Template).
Do not change the volume of any of the reactions. All components have been optimized for the
volumes specified.
1. Prepare enough of the following Buffer Mix for all of the 5’- and 3’-RACE-Ready cDNA synthesis
reactions plus 1 extra reaction to ensure sufficient volume. Mix the following reagents and spin
briefly in a microcentrifuge, then set aside at room temperature until Step 6: 4.0 µl 5X First-Strand Buffer
0.5 µl DTT (100 mM)
1.0 µl dNTPs (20 mM)
5.5 µl Total Volume
2. Combine the following reagents in separate microcentrifuge tubes:
For preparation of
5’-RACE-Ready cDNA
1.0–10 µl RNA*
1.0 µl 5’-CDS Primer A
0–9 µl Sterile H2O
11 µl Total Volume For preparation of 3’-RACE-Ready cDNA 1.0–11 µl RNA* 1.0 µl 3’-CDS Primer A 0–10 µl Sterile H2O 12 µl Total Volume
*For the control reactions, use 1 µl of Control Mouse Heart Total RNA (1 µg/µl).
3. Mix contents and spin the tubes briefly in a microcentrifuge.