Hello, I have a following issue: I extracted approx. 20 plant DNA samples from dry leaves (old herbarium items) using Qiagen DNeasy Plant Mini kit. When I run a PCR for housekeeping gene (alpha tubulin) for these samples, they are all amplified and nicely visible on the agarose.
However, when I try to use ISSR markers for those same samples, almost all of them fail repeatedly - no amplification, no bands on gel. I was thinking this could be due to DNA fragmentation because the samples were old, but I know that it is possible to successfully analyze even older DNA (just look at archaeology for example, although the methods and technology there is probably top notch and incomparable to what I have, that si true ...).
So I still don't know where the issue is, or how to figure this out. I believe changing the annealing temperature is out of question, because that could lead to unspecific bands and later during matrix construction samples would be incomparable to the rest in the set (the other 50 samples work normally, were collected and dried more recently). I still use the same polymerase and it works for other samples. Do you think that playing around with template concentration could do something in this case ?
Thank you very much.
Do you have a good positive control sample? Test out your primers using the positive control and a no template control using gradient PCR. You might need to optimize the annealing temperature.
I agree with Katie A Burnette. Because you did have a good result with housekeeping gene indicating your DNA is still good for PCR. However, for SSR or ISSR makers, the DNA quality is very important to have a success. There are some steps you may perform to optimize your experiments.
- You may use CTAB method for DNA extraction. Dry leaves are not good for Plant Mini kit at some cases.
- Measure your total DNA by nanodrops and perform gel-running using agarose 1% to check your DNA quality and concentration, then dilute to 50-70 ng/micro L
- Test your ISSR primers using a good DNA (positive control) to check primer quality and also find a good condition for PCR
- As you primers and DNA are good enough, you should perform ISSR-PCR for all your samples
Hope it helps
Dear Petel
I agree with Phat concerning the DNA extraction method. A CTAB based method for many plant materials far better than the kit you used. We do not know which species you are working with. It might be that you need to further purify the DNA extract. In order to avoid losses of DNA during extra purifications, try the QuantaBio ToughMixes (Perfecta ToughMix or other that the commercial may recommend to you). Incredible results may be obtained. Anyhow, you would need to optimise your PCR conditions for this new mix.
Good luck.
Katie A Burnette Phat Tien Do Eugénia De Andrade Silva Perhaps I am quite late, but I just wanted to thank You all for Your valuable insights. I´ll see what can be done with the samples.
All the best.
Plant tissue culture (micropropagation) is recognized as one of the key areas of biotechnology because of its potential use to regenerate elites, while conserving valuable plant genetic resources. However, the scaling up of any micropropagation methodology carries the risk of inducing genetic variability, namely somaclonal variation among sub-clones of one parental line.
Ozodbek there many ways to check genetic stability of the micropropagated plants. You can use dna or phytochemical markers, or you can also analyse the size of genome of the mother and in vitro clones using flow cytometry.
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