I am doing RAPD analysis to test for clonality in wild populations of three different plant species (Trientalis europaea, Cornus suecica and Rubus saxatilis). While I get quite good results for one of the species (Trientalis europaea), amplification fails for the other two.
I extracted DNA with the Qiagen DNeasy Plant Mini Kit and I tested 60 decamer operon primers of three different series (A, N and AF). I already optimised my PCR protocol in many ways (annealing temperature/number of cycles/PCR program, type of polymerase, amount of Mg, amount of DNA etc.), but without any success. Besides, since I had successful amplification for at least one of my species, the problem seems to be species-specific rather than protocol related.
Attached is a gel photo of my latest run which shows failed amplification for one of my focal species while my positive controls show clear bands, which suggests the problem is not due to PCR conditions. What am I doing wrong or overlooking?
I would very much appreciate any help.
I guess you are using different components of PCR rather than ready-made master mix. In that case, you need to check each and every component (eg - DNTP, Taq, etc., sometimes the quality may deteriorate when we continuously freeze and thaw them. Same goes with the primers also).
So, I would recommend to do it from the first step.
1. Isolate DNA by CTAB method (reference at the end).
2. Check if your primers are ok. If so, then go for gradient PCR and find the annealing temp.
3. If possible, use ready-to-load ready made master mix. It not only saves time, but also increases the efficiency.
Reference:
Allen, G. C., Flores-Vergara, M. A., Krasynanski, S., Kumar, S., & Thompson, W. F. (2006). A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature protocols, 1(5), 2320-2325.
PS: Since the positive control is showing clear bands, so the DNA is ok. Try increasing the quantity of DNA and also try with a slight increased conc of EtBr in the gel. And recheck your annealing temp by using gradient PCR.
Thank you all very much for your suggestions.
I agree with Mostafa Farajpour that something in the DNA must inhibit amplification. I assumed DNA extraction kits (spin columns) were already optimised for removing phenolic compounds, compared to for example CTAB. However, there is no separate step included to remove phenols in the protocol of the DNeasy Plant Mini Kit. There is such a step in the protocol of the DNeasy Plant Pro Kit, but I was unsuccessful in getting good quality DNA using this kit (DNA looked degraded on gel).
For your convenience, I included both protocols in the attachments. Note however, that the manufacturer (Qiagen) does not provide the contents of any of the reagents.
Is there a way to get rid of phenolic compounds after extraction? This would safe me time and I do not have enough starting material left for all of my samples.
Hi Lilian Seip
Our CLIQS 1D Pro software is designed to perform this kind of analysis, you can get a free trial from here: https://totallab.com/products/cliqs-1d-pro/
Best wishes,
Steven
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