I am preparing to use some plasmid DNA to spike into soil samples at a known concentration as an internal standard to account for variable DNA extraction efficiency. I will also be looking at bacterial community composition (Illumina) and relative abundance (qPCR) using general 16s primers in these samples, so I am concerned about the possibility of adding E. coli 16s DNA along with the plasmid. My plasmid mini prep looks great on the gel and shows no sign of genomic contamination, but I am wondering if there is likely to still be enough bacterial genomic DNA to amplify? Does anyone know of a way to completely remove all traces of genomic DNA from a plasmid prep? Maybe I am being overly paranoid...
No, you are not too paranoid as there will usually be enough traces of genomic DNA to create problems. One way to get rid of that is treating your plasmid DNA with exonuclease which will remove linear DNA. But you should always check afterwards by PCR if the removal was indeed complete.
Thanks for the reply. One possibility could be to use PCR product instead of plasmid DNA as the internal standard to be spiked into my soil samples. This would avoid the potential for bacterial 16s contamination in the mini prep. But, I am unsure if this is an acceptable approach. Would a 600 bp amplicon be significantly less stable than a plasmid throughout a typical soil DNA extraction protocol (I am using the MO BIO soil kits)? My original decision to use plasmid DNA as the standard was based on a few papers I read, but they were primarily concerned with soil fungi and therefore did not have to worry much about a little bacterial genomic contamination. Using purified PCR product would actually be much easier for me, but I wonder if anyone can see a reason not to go that route?
If the MO BIO soil kit includes a decent DNAse inhibitor, you should be able to use any spike you wish. A double-stranded DNA PCR product would be a great choice for you here - for the reasons you've stated. If the kit does not provide a way to stop DNAse activity in your soil samples, a nice post on several possible ways to do that that can be found here:
http://www.researchgate.net/post/What_is_the_best_way_to_inhibit_DNAseI
Stopping DNA degradation as soon as possible in your samples/extracts would seem to be the biggest concern. Differing degrees of nuclease activity from sample to sample could really throw your results off and could also be misconstrued as variable extraction efficiency - but is really differential DNAse degradation of target sample DNA and spiked DNA. For RNA there are well-known RNAse inhiobitors (RNaseOUT, RNAsein, etc.), but it is generally harder to find DNase inhibitors of the same kind, although various antibodies against DNA nucleases have been proposed as/in potential patents over recent years.
- Badges
- Science topic