I performed PCR which targets bacterial 16S (using GC968f/1401r) and fungal 18S (using NS1/GCFung), each produced ~400 bp and ~350 bp, receptively. The DNA was extracted from soil sample.
Can anyone suggest the amount of PCR amplicons (ng) that can be loaded per lane for DGGE?
Dear Muhammad,
You can find some hints about DGGE in this website http://ddgehelp.blogspot.pt/2005/11/dgge-guide.html and in this chapter http://www.intechopen.com/books/gel-electrophoresis-principles-and-basics/denaturing-gradient-gel-electrophoresis-dgge-in-microbial-ecology-insights-from-freshwaters
We usually load ~700ng. But I think you should make some tests and adapt to your samples.
Hope it will help.
Regards,
Isabel
nice question I read all comments and know what u mean
thanx and good luck
The best way to know it is to make some tests, as Isabel told you. I think that 700 ng is kind of excesive, I worked with about 150 ng (my sample came from mine tailings), what kind of soil are working with?.
What do you want it for? Are going to sequence the bands? That's another important thing to decide the amount of PCR amplicons. Avoid using to much DNA because your lanes could end full of bands.
Regards.
Mario.
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