This is likely to be a very basic question - I am sorry for asking it but will do that anyway:)
We are trying to clone and sequence bacterial communities coming from different samples by using the same chemistry (well, mostly kits...) The total DNA concentration extracted from the samples is not predictive in any way of the number of clones growing on the plates. Moreover, some high-DNA samples produce notoriously low number of clones (blue-white screening) and have a poor sequencing success (16S rRNA) compared to other samples. What would be a plausible explanation for that (apart from a high contribution of non-bacterial DNA to the total sample DNA)?
There are a few possible explanations that might be worth considering. First, your determination of DNA concentration might be incorrect. If you are using absorbance at 260 as your measure then having RNA contamination or small oligonucleotide contamination (stemming from degraded DNA) this could you give you an erroneously high DNA concentration. Secondly, it might also be possible that your DNA is not sufficiently purified and there are some contaminants that are either degrading your sample or otherwise inhibiting your subsequent reactions.
Yes, both are possible, and I like this line of reasoning... But this would be true for all DNA samples, wouldn't it? In our case, we collect biofilm from different substrates and some substrate types consistently produce samples that, despite their higher DNA content, produce very few clones, and even fewer those amenable to sequencing... all DNA samples are purified and have excellent 260/230 and 260/280 ratios.
Have you compared the DNA samples on a gel. You can have DNA that has excellent 260 ratios but is still degraded.
Why would we get degraded DNA time after time from one substrate but not the others?
No, we didn't compare extracted DNA, but PCR product for just these samples is indeed different (more smear, less clear bands - still should be OK for cloning, I think).
Elena,
smeary= degraded = not good for cloning.
If you get smears after PCR may be your DNA is being degraded after purification. It would be a good idea to check it in agarose gels.
Thank you Estanis. Yes, We fully realize that poor PCR product is not good for cloning. In this case, my question is rather more general - why samples of bacteria collected from some substrates in sufficient quantities (by total DNA) may produce this kind of poor PCR product and poor clones? I can also add, perhaps, that this was an experiment where different substrates were provided to the same bacteria stock for biofilm building.
Elena, I'd bet that your problem is with the quality of the DNA prep. May be you have a "carry-over" of some contaminant/s that inhibit downstream enzymes, e.g. carbohydrates are notorius inhibitors of restriction enzymes. If possible I would try with a diferent purification method.
This could have happened, of course, but - again - if we do 20-25 independent samples from substrate X and the same number from substrate Y using the same extraction and purification methods, then why only those coming from Y are having this problem?
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