SMARTer_RACE_5_3__Kit_User_Manual_022714(16)

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.

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