Hello
I try to amplify gene of interest by RACE-PCR. For 5` RACE, I saw multiple bands after using specific nested primer. But The desired band was not seen on the gel. What is the reason?
When I loaded PCR product without nested PCR, I saw fade band that is true for my work. But I could not recover it for cloning because of disappearing after running agarose gel. What`s the solution?
Regards
How much first round pcr product are you re amplifying and for how many cycles with the nested primers?
Bands fading usually means that the gel has been on the UV lightbox for too long and the signal fades. Take a picture quickly when you see the result. A band of the right size is good but not necessarily the correct band.
It is very easy to over amplify a pcr product and just end up with a long smear of amplified material if you use too much first round product as template or too many cycles of pcr. Try 1ul of 1/100 dilution of the first round product and 15-20 cycles of pcr to get bands rather than an over amplified smear.. If there is no visible amplimer at all then you should run a temperature gradient pcr to see what annealing temperature works for your nested reaction.
Possible reasons for not seeing the right size bands are....the sample is not expressing the gene, the primers are not specific for the gene or are badly designed and the annealing temperature of the pcr is too high for the primers to work.
I don’t know what kit you are using. I have used “FirstChoice® RLM-RACE Kit”.
You should try to increase your target concentration:
First of all, it is essential that the starting RNA be of the best possible quality.
Since there are several treatments to remove free 5'-phosphates and cap structure from 5' ends of mRNA, you should be careful about these steps to do accurately. Also, remove all traces of treatments properly (e.g., using phenol/chloroform extraction and ethanol precipitation), so 5' RACE adapter ligation to decapped mRNA will be done properly.
Another point regarding the nested PCR step: in general, the annealing temperature in the outer PCR is less critical. The annealing temperature of the inner, nested PCR may need to be higher than predicted by calculation or by primer design software to achieve the required selectivity in the amplification.
However, in your case, you should lower the annealing temperature to get the desired band sharper.
Moreover, the lack of a specific RLM-RACE product may be dependent on the distance between your nested primers and the 5' end of the target.
For more information please refer to the attached file:
which kit did you use? Try to change PCR program ( or annealing temp).
OR use Invitrogen RACE PCR kit for RACE PCR amplification. it works well.
Paul Rutland Thank you so much. I checked all of things you explained except gradient PCR. I will try it.
Shirin Parizad Thank you so much. I will try upper annealing temperature for 5`nested PCR.
Atia Gohar Thank you for your recommendation.
Dear Zahra Sadat Eslami, You're welcome.
But I think there is a misunderstanding ...in my second answer, I said the general way of doing nested PCR and for having multiple bands you should go for increasing the annealing temperature. However, in your case, it doesn't matter having a few bands without the desired one, so you should "decrease" the annealing temperature to get a sharper desired band.
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