clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual
3. Commence thermal cycling using one of the following PCR programs (both programs 1 and 2 work
with the positive control 5’- and 3’-RACE TFR and UPM Primers). Be sure to choose the correct
number of cycles (as noted) based on whether you started with poly A+ or total RNA. NOTES ON CYCLING: You may need to determine the optimal cycling parameters for your gene
empirically, because the number of cycles necessary depends on the abundance of the target
transcript. Run 20 or 25 PCR cycles first as described and analyze 5 µl from each tube, along with
appropriate DNA size markers, on a 1.2% agarose/EtBr gel. If you see weak bands or no bands, return
the tube(s) to your thermal cycler and perform five additional cycles (according to the third set of cycles for touchdown PCR). The optimal extension time depends on the length of the desired
amplicon. For 0.2–2 kb amplicons, we typically extend for 2 minutes; for 2–4 kb amplicons, we
extend for 3 minutes; and for 5–10 kb amplicons, we extend for up to 10 minutes.
NOTE: The Tm should be calculated based upon the 3’ (gene-specific) end of the primer, and NOT
the entire primer.
Program 1 (touchdown PCR—preferred; use if GSP Tm >70°C)
5 cycles:
94°C 30 sec
72°C 3 min*
5 cycles:
94°C 30 sec
70°C 30 sec
72°C 3 min*
20 cycles (Poly A+ RNA) OR 25 cycles (Total RNA):
94°C 30 sec
68°C 30 sec
72°C 3 min*
*If fragments >3 kb are expected, add 1 minute for each additional 1 kb.
Program 2 (use if GSP Tm = 60–70°C)
20 cycles (Poly A+ RNA) OR 25 cycles (Total RNA):
94°C 30 sec
68°C 30 sec
72°C 3 min*
*If fragments >3 kb are expected, add 1 minute for each additional 1 kb.