I am performing RACE PCR and in experiment of 5' and 3' PCR with the UPM and gene specific primer.... I m getting numerous band. Normally we should not get any band in the gel. I do have isolated RNA and then checked the RNA integrity... it showed me perfect. But I don't understand why I am getting numerous bands... U suggestions will help me to troubleshoot.
Thanks in advance.
Numerous bands could be caused by an unspecific binding of your gene specific primer. If you are working with eukaryotic RNAs, alternative splicing could explain some bands. You should expect at least one band in the gel. Why are you surprised by having a PCR product at all? If there are too many PCR products, the conditions of the PCR could be adjusted. A higher annealing temperature might help.
Actually sir, I am doing RACE PCR in which I am using a gene specific primer for 5'and 3' along with UPM.... In which the bands should not be seen.
I have also faced the same problem but after optimizing PCR conditions like Ta, cycle etc. it comes fine.
okkkk...... but this was the repetition which I was doing. For the first time when I have done with the same PCR conditions... it did not show any band... but when I am repeating it is showing this type of result. What can be the problem then.
I have used SMARTer® RACE 5’/3’ Kit User Manual and manufacturer protocol given were
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(ta) 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(ta) 30 sec 72°C 3 min*
I have used both protocol depending upon the gene. For my case the problem was resolved by lowering Ta to 64-65 °C.
Good luck.
Hi Rajahan, check properly because the manual says using gsp and upm must give bands but if you do a negative control of gsp only and upm only you should not been seeing any bands. check that becasue thats what my manual says.
PCR reaction consisting UPM with GSP will produce your "transcript of interest". Getting one or more than one band is usual during the RACE. The multiple bands in the 5' RACE can be explained as
multiple transcripts/single with multiple transcription initiation sites/ truncated cDNA due to thr RNA degradation and finally may be due to the non specific binding of your gene specific primer. Try different Tm during the Nested PCR.
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