Hi, I want to ask what is DGGE marker used for? Since there almost no commercial DGGE marker. I can make my own marker by myself, but does the marker make any sense when you analyze the DGGE gel ?
Thanks！
A "DGGE marker" can be helpful if you need to compare DGGE profiles of samples run on more than one DGGE gel.
As you probably know, DGGE profiles of samples are best compared if the samples are run on the same DGGE gel, since conditions between different DGGE gels are not completely identical. However, having a "marker" will allow you to align to some extent DGGE profiles obtained from different DGGE runs.
One possibility is to run one of your samples and excise bands from your gel.
You could also identify bacteria with different GC content in the region you amplify and get them from a culture collection.
Akadiri Olalekan Oladimeji , can you be a little bit more specific? Do you want to analyze an image of a DGGE gel?
Thank you @Wolf T Pecher. I actually have challenges in getting my DGGE to work. The ladder I used showed but the sample did not. I have attached the PCR Gel picture and the DGGE gel picture here
Akadiri Olalekan Oladimeji , it looks like to me that you may have not enough pcr product to start with. There are also many other questions, f.e., what is your size of the PCR product, what is your gradient, did you use a GC clamp. etc.
A good resource to look at is a guide by Stefan Green. You can access it through the blog: http://ddgehelp.blogspot.com/
@Wolf T Pecher Thank you so much for your helpful reply. I used a GC clamp 16sRNA primer. The size is at about 500bp which I think is correct on AGE. But like you rightly pointed out I think the faint band on the AGE is a prove that I may not have enough PCR product
I will try to optimise the PCR process and try again. Thank you Sir
Akadiri Olalekan Oladimeji Indeed, may use a nested PCR approach. The guide I mentioned was very helpful to me when I started with DGGE. Once you have worked out the conditions DGGE works like a charm.
I am still not getting it right ..... I already optimised PCR with another master mix. I got a clearer band on AGE. But my DGGE still turned out to be aweful. I made a 6% gel with 30%-60% gradient. All buffer and reagents used are freshly prepared with molecular grade reagents too. What could I be doing wrongly?
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