Is there anything I can do to reduce the amount of fuzzy and extra bands that appear behind my DGGE? Its making it difficult to differntiate between the bands I need. The standards are located at the ends and middle and should have 6 bands which I can identify, however, the other bands I do not now what shouldn't. Could this be a result of PCR messing up?
It's a 6% acrylimide solution with a stacking gel. Runs in 0.5xTAE for 16 hours.
The gel run itself may not be your problem, unless there is considerable temperature differences during the run and different portions of the gel are at different temperatures.
I would suggest re-optimizing conditions of PCR, failing that, synthesizing different primers, if at all possible.
I think also that there will be a problem with the PCR. Your standards are contaminated I think. It can be that when you load the gel, some of the sample are not loaded perfectly and then contaminate the samples next to it. It's just strange then that the standard at the left has the same contamination as the other ones. So I think its the PCR that has to be optimalized cause the DGGE looks very fine to me.
For the PCR, you can try to use a higher anealing temperature so te primers are more specefic, and if that doesn't work you maybe have to use other primers.
I also see that you have a lot of bands so it will be for sure difficult to analyse the samples! I have the same problem with bacteria in soil and it was very hard.
the image looks for me that you have a problem in the PCR. The double bands in your standards seem to be the result, that your PCR product don´t have the same lenght. what I did was that I Put the whole PCR product on a normal gel and than cut the distinct band eluet the DNA and use that for the DGGE.
Within info about the type of sample and the genomic region amplified and primers used is not easy to concluded many things. As it's mentioned before, the constant temperature across the gel is important, also let the gel to complete a full polimerization.
I guess you check your PCR product in agarose before DGGE, maybe yoy can purify the prduct before DGGE.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques