Hi everyone.
I'm trying to extract DNA from Gram-negative bacteria through a metabolic process, and using the GeneElute Bacterial Genomic DNA extraction kit (Sigma)
My set of samples covers the entire growth curve. At the beginning of the process, during the exponential growth phase, I don't have any problem and although sometimes there is some degradation, I can observe a nice chromosomic band at the top of the gel when running agarose gel electrophoresis. The problem is when the process is more advanced, because the bacterial culture enters into the stationary growth phase. At this stage, DNA appears to be degraded.
Does any one any tip to improve the quality of the material obtained?
I attached an image of the last extraction
Well, why would you want to extract genomic DNA in the stationary phase to begin with? If the culture is pure, then the DNA obtained would be the same, and you can choose the best culture-time to yield the best DNA quantity...
Nevertheless, the loss of DNA from the stationary phase could be a problem related to the ratio of viable cells vs. dead bacterial cells... Assuming your going with a fixed protocol from the kit, then you're probably using a given quantity (lets call it x) of starting sample to the DNA extraction. The quantity x of bacterial culture when sampled from an exponencial-growth culture will contain much more living cells (=intact DNA) than that same quantity x of stationary phase, where you can have a maximum of bacterial cells in the whole petri dish, but where the amount x carries much more degraded biostuff.
If insisting in obtaining DNA from the stationary phase, I would increase the amount of sample introduced in the kit, lets say, to 3x (I guess it will not change much in terms of volume), and see if it fixes the problem.
You should get ample genomic DNA at any stage. I never used any kit for genomic DNA extraction. The degraded or lack of DNA in your sample could be a technical issue rather than the nature of growth phase. I hope your buffers are good with no DNAse contamination. Here is what I do to extract genomic DNA:
1. P1 buffer for plasmid extraction containing RNAse is my initial buffer to resuspend cells in. I would then add 10% SDS (10-20microL in 100microL volume). Mix by tapping (do not do vigorous mixing at any stage).
2. Incubate at 65 for 30 minutes (1 hour is OK as well).
3. Add Phenol-chloroform mix (equilibrated) equal amount (100microL for 100microL sample). Mix by tapping (no vortexing).
4. Spin at high speed for 10 minutes.
5. Take the upper aqueous phase. Add 100% ethanol to precipitate out the DNA (500microL in 100microL sample).
6. At this point, you can see the genomic DNA precipitating out. Spin at high speed for 1 min.
7. Throw the supernatant and wash the pellet with 70% ethanol.
8. Air dry and dissolve in TE buffer. Your genomic DNA is ready.
Best of Luck.
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