It has been suggested to me that microsatellite data collected on so-called ´manual´ polyacrylamide gels - with silver-staining - is generally not acceptable to the majority of journals now. We have a couple of projects starting up on limited budgets, and are thinking of using manual gels. Surely if the data are collected in a controlled and systematic manner, a MS cannot be rejected solely based on detection technique. What experiences have others had?
I am also genotyping fungus using SSR markers with PAGE Gels. no Doubt the detection technique is no sensitive as fluorescent based one (Fragment analyzers like LICOR or BIO ANALYZER 2100) and you will not get much alleles as compared to later one. I think the paper can not be rejected based on silver staining (Which is better than Agrose gel) however analysis of data should be done using recent updated freely software's. The manuscript are rejected as they lack novelty and their discussion and introduction. Both these are very important as for as manuscript is considered.
We use silver staining. The decisive factor is the level of data processing.
The Gel techniques are not nearly sensitive enough, you could miss alleles if the amplification is not high enough, or if alleles are almost identical. The manuscript may not be rejected but more data might be needed to factor out human error and differences in run time and such. I would suggest partnering with an institution that could help in this regard and maybe cut costs. Software packages such as GeneMarker and PowerMarker allow for more applications of data obtained from BIO ANALYZER or other computer-based sequencing.
I have used silver staining and also SybrGold to stain my PAGE gels. The SybrGold was far more efficient and sensitive, without the mess and time of silver staining, and without worrying about over exposure. We simply added 1ul of SybrGold to 10ml of ddH2O, spread this over the gel and left it in darkness for around 30mins. The tricky part is visualizing, and depending on your GelDoc this could get really fiddly... let me know if you want details! (if you have the money you could just buy non-UV absorbing glass for your plates and then this step becomes a lot easier)
I recommend you call alleles with two independent researchers - and do plentiful positive controls (repeats on different gels) and negative controls. With very good quality control you can better justify your technique. And I agree with Jordan - SybrGold is a good choice - we did this same thing with an agarose overlay - mix sybr into agarose (1 ul to 10 ml of 1% agarose/TBE or H2O) and pour over gel... we used legos to make a frame to pour it in... this works really well if you have the non-UV absorbing glass - no wet mess to deal with.
Many thanks everyone for your answers. We continue to explore options, including collaborations to run the fragments automatically. Jordan and Amy, I may well get back to you both for further details on your protocols, if that´s ok. If you can point me to any literature or online resources in the meantime that´d be great.
Silver staining shows not specific bands in polyacrylamide, agarose gels is more dificult to separate bands with few base pairs differences, I don´t work with SybGreen with SSR and I don´t know if is better to distinguish not specific bands.
Happy to help Nicola, just let me know if I can help. I will see what I can find for you...
I agree with Padder that MS can not be rejected based on the technique used for SSR marker profiling on PAGE. Mostly the papers are accepted for novelty and a sound discussion. I do agree that the analysis of SSR markers on PAGE by silver staining has some limitations viz., high sensitivity which detects non-specific amplifications, laborious experimental work of analyzing large number of samples on the gel and data recording etc. However, the techniques remains with in the reach of many researchers with limited funding at their disposal.
The major problem doing on PAGE by silver staining is the laborious experimental work, the health hazards associated with it and the painstaking scoring. I suggest that you have both positive controls and negative controls and also see to it the manual scoring is rechecked independently by another researcher
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