clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual
VI. Rapid Amplification of cDNA Ends (RACE)
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING.
At this point, you have 3’- and 5’-RACE-Ready cDNA samples. The RACE reactions in this section use only a fraction of this material for each RNA of interest. There is sufficient single-stranded cDNA for PCR amplification of multiple genes.
A. Things You Should Know Before Starting RACE PCR Reactions
If you intend to use LD PCR to construct your full-length cDNA after completing 5’- and 3’-RACE, be
sure to set aside an aliquot of the 5’-RACE-Ready cDNA to use as a template in the PCR reaction.
Please note that the efficiency of RACE PCR depends on the abundance of the mRNA of interest in your
RNA sample. Additionally, different primers will have different optimal annealing/extension
temperatures. Refer to the Troubleshooting Guide (Appendix A) for suggestions on optimizing PCR conditions.
NOTE: This is a RACE-specific protocol. It differs from the general SeqAmp protocol in many regards.
B. Protocol: Rapid Amplification of cDNA Ends (RACE)
This procedure describes the 5’-RACE and 3’-RACE PCR reactions that generate the 5’ and 3’ cDNA
fragments. We recommend that you also perform positive control 5’- and 3’-RACE using the TFR
primers and UPM. Although the Universal Primer Short (UPM short) is provided, nested PCR is
generally not necessary in SMARTer RACE reactions.
1. Prepare enough PCR Master Mix for all of the PCR reactions plus one extra reaction to ensure
sufficient volume. The same Master Mix can be used for both 5’- and 3’-RACE reactions. For each
50 µl PCR reaction, mix the following reagents:
15.5 µl PCR-Grade H2O
25.0 µl 2X SeqAmp Buffer
1.0 µl SeqAmp DNA Polymerase
41.5 µl Total Volume
2. Prepare PCR reactions as shown below in Table 2. Add the components to 0.5 ml PCR tubes in the
order shown and mix gently.
Table 2. Setting up 5'- and 3'-RACE PCR Reactions
Component
5’- or 3’-RACE-Ready cDNA
(experimental)
10X UPM
5’ or 3’ GSP (10 µM)
H2O
Master Mix (Step 1)
Total Volume 5’- or 3’-RACE Sample 2.5 µl 5 µl 1 µl — 41.5 µl 50 µl UPM only (– control) 2.5 µl 5 µl — 1 µl 41.5 µl 50 µl GSP only (– control) 2.5 µl — 1 µl 5 µl 41.5 µl 50 µl