I'm conducting research on endophytic microbial community. I'm targeting 16S analyses but I realize the problem of chloroplast 16S . Though the last resort to avoid sequencing chloroplast 16S is through selecting hypervariable regions specific for bacteria (i.e. V5-V7, although these regions have lower scope of bacteria according to Silva Database), I have been doing chloroplast removal approaches prior to sequencing.
So far, I have tried using semi-protocols for chloroplast isolation using centrifugation and buffers containing sorbitol. But no good result since the density of chloroplast is only a little higher than bacterial cells. Is there any way I can make them precipitate, like making them heavier using chlorophyll-binding substance?
THE separation of some plant cell components is complicated by the presence of dispersed chloroplast fragments. Several procedures for removing these are used; they are based on the denaturation of these fragments, and include heating, precipitation by acids or alcohols, emulgation with chlorocarbons and (or) fluorocarbons and adsorption techniques. All these methods facilitate the sedimentation of the chlorophyll containing material to the bottom of the centrifuge tube or at the interphase-between denser solvent and water layer in the form of a large cake. Such treatments, in addition to the variable denaturation of soluble proteins, cause serious if not entire loss of the minor cell components as a result of co-precipitation.
Dear Dale Pinili,
I think the attached papers would be helpful while clearing your doubts.
We have this problem with a current algal microbiome project and had to design PNA blockers for the host chloroplast genes. This doesn't answer your specific methods question, but is a solution to consider if you have limited host species. PNAbio actually sells PNAs that were designed for higher plants; reported by Lundberg et al. in 2013; Nature Methods.
http://www.nature.com/nmeth/journal/v10/n10/abs/nmeth.2634.html
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