Im performing an experiment where i'm trying to isolate microbes from an insect gut. In other papers that I have looked up, they used the QIA amp DNA mini kit. In the kit handbook, there is a protocol for isolation of bacteria and another protocol for tissue isolation.
Which method is best for isolation of bacterial DNA from insect tissue using QIAamp DNA kit by Qiagen? tissue isolation, or bacterial isolation?
I would greatly appreciate any help!
Dear Sam, you can isolate bacteria using selective media if you have a target bacteria to isolate, or after enrichment you can isolate total bacterial DNA from insect surfaces, then by Real-Time PCR or using genus or species specific primers by conventional PCR, you can examine the presence of specific bacteria.
Dear Sam, I agree with the previous answers. However, the choice of method will depend upon the experimental design and the research problem being investigated. If you simply wish to study the cultivable gut bacteria, then you can go for bacterial enrichment and isolation method, mentioned by Ali and Mert. This method will give you bacterial isolates from where you can isolate DNA which is free of host DNA. Use this DNA for PCR amplification of 16S gene using universal primer, clone, sequence and do BLAST analysis of the sequence data. The limitation of this method is that you will only be able to isolate and sequence a very few of the gut bacteria, since most of the bacteria present in the gut are uncultivable on common media.
Alternatively, if your research objective is to study the total bacterial population (cultivable as well as uncultivable), then you have two options. You have to dissect out insect gut (if possible), then isolate total DNA, enrich the bacterial DNA using RCA based method (this will selectively amplify circular DNA) and go for whole genome sequencing using any parallel sequencing platform. Or you can use the total DNA extracts for PCR using bacterial universal 16S primers and then go for bacterial V3-V4 based metagenomic approach, which is relatively much less expensive than whole genome sequencing. So, wisely decide the method depending upon your actual query.
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