How can I interpret the presence of smear on my agarose gel after extraction of gDNA from sample of cow dung and manure? I have good absorbance as well as for A260/280 and A260/230 ratio. My DNA concentration are between 20 and 30ng/ul. However, on the agarose gel, smear are observed on all samples. Does this have an impact on the quality of my DNA extracts (denaturation)? Can I make a qPCR on it or not?
There will always be shearing when dealing with gDNA unless the starting cells are embedded on agarose plugs and digested in situ. What matters is whether the amount of shearing interferes with the downstream assay. In this case it is OK to do qPCR, as the size of the amplicons is much smaller than the modal size of your gDNA fragments.
Hi Mamadou Camara,
is it possible that the smear on the agarose gel comes from the electrophoresis conditions you applied. The buffer, the agarose concetration (< 1%), the applied voltage (40-50 mV, 1.5 h), and the amount of loaded gDNA effects the tension of the gDNA through the gel. The DNA bands of your DNA ladder looks like horns of a bull what is in most cases an argument for harsh electrophoresis conditions. I guess if you apply the 'softer' conditions you will get better results. Additionally, you can increase the resolution, when using 2/3 of the agarose gel length instead of the 2 cm. All the Best!
Thank you for your suggestions, I'll take account of it.
Nevertheless, seen the states of my total DNA extracts and the fact that the primers that i will use are designed to amplify rDNA 16s from 150 to 500bp?Does it is always a matter to do qPCR? I saw in the littarature that those primers are frequently used (Yu et al. 2005).
Your DNA quality is what can be expected from such extractions and is of the same DNA quality in the precise paper you're refering to (Development and Application of Real-Time PCR Assays for Quantification of Genes Encoding Tetracycline Resistance in AEM i suppose) ?
To answer precisely your question: I've been using these with similar samples origin (soil, feces), extracted by bead-beating (with most fragments size around 20kb and relatively similar smearing as shown in your photograph, observed on gel, yet, I do not know how much is loaded on your gel, and results were satisfying with qPCR, metagenomic sequencing, and all downstream applications. No cloning experiments were performed though, so I can not comment on that.
I'd also add that DNA extracted from environmental samples will always yield a consequent share of dead bacteria, not maintaining their genomes and hence already sheared, prior to extraction.This is also inversely proportionnal to your samples bilogical activity, hence, rich biomass samples with more active bacteria in relative abundance will show less smearing, for the same DNA mass migrated on gel.
However, even when resolving high-molecular wieght DNA from environmental samples using agarose-plugs lysis and pulsed-field gel electrophoresis migration, there is always a significant share of smearing.
For reference on extracting DNA high-molecular weight (>40Kbp) from environmental samples with high quality yielding satisfactory cosmid / fosmid / BAC cloning, I'd suggest this reading, which is the above mentionned protocol paper:
1. Bertrand, H., Poly, F., Van, V., Lombard, N., Nalin, R., Vogel, T., and Simonet, P. (2005). High molecular weight DNA recovery from soils prerequisite for biotechnological metagenomic library construction. J Microbiol Methods 62, 1–11.
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