clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual
4. [OPTIONAL] If the primary PCR reaction fails to give the distinct band(s) of interest or produces a
smear, you may wish to perform a secondary, or “nested” PCR reaction using the Universal Primer
Short (UPM short; supplied) and a NGSP (See the discussion in Section IV.D.) This result is more
common for transcripts that are less abundant. The suggested secondary PCR will most likely result
in the expected distinct band(s).
a. Dilute 5 µl of the primary PCR product into 245 µl of Tricine-EDTA buffer.
b. Repeat Steps 1–3 above, using:
i. 5 µl of the diluted primary PCR product in place of the RACE-Ready cDNAs.
ii. 1 µl of the Universal Primer Short and 1 µl of your nested GSPs. iii. 15–20 cycles of Program 2.
NOTE: The Troubleshooting Guide (Appendix A) discusses several control reactions that will
help you troubleshoot your RACE reactions if yields are suboptimal.
VII. Characterization of RACE Products
At this point, we recommend that you characterize your RACE fragments and confirm that you have amplified the desired product. This procedure can prevent confusion and wasted effort when you generate the full-length
cDNA, even if you have single major products from both the 5’- and 3’-RACE reactions. Characterization is
especially important if you have multiple bands or if you suspect that you are working with a member of a
multi-gene family. Multiple bands are more common with 5’-RACE than with 3’-RACE. Multiple transcriptional start sites tend to create a number of different transcripts, and there’s a good chance these multiple bands are real variants and not artifacts.
We provide you with the materials necessary for the suggested method of characterizing RACE products via cloning and sequencing (Sections B & C, below).
A. Protocol: Gel Extraction with the NuceloSpin Gel and PCR Clean-Up Kit
For more details on the included NucleoSpin Gel and PCR Clean-Up Kit, please download its User
Manual from our website at .
Before you start: Add 24 ml of 96–100% ethanol to Wash Buffer NT3. Mark the label of the bottle to
indicate that ethanol was added. Wash Buffer NT3 is stable at room temperature (18–25°C) for at least
one year.
1. Electrophorese your RACE DNA sample on an agarose/EtBr gel. We recommend using a buffer
system containing either TAE (40 mM Tris-acetate [pH 8], 1 mM EDTA) or TBE (45 mM Tris-borate [pH 8], 1 mM EDTA).
2. Locate the position of your fragment under UV light. Use a clean scalpel or razor blade to excise the
DNA fragment of interest. Cut close to the fragment to minimize the surrounding agarose. Estimate
the amount of DNA present in the gel slice.
NOTE: Minimize UV exposure time to avoid damaging the DNA.
3. Measure the weight of the gel slice and transfer it to a clean 1.5 ml microcentrifuge tube.
4. For each 100 mg of agarose, add 200 µl Buffer NTI.
5. Incubate the sample for 5–10 minutes at 50°C. Vortex every 2–3 minutes until the gel slice is
completely dissolved.