Stool Microbial DNA Isolation: Studying the Effect of Environmental Enrichment on Microbiome Diversity in Murine Tumor Model

Abstract

Source: Fuller, A. K, et al.  A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model. J. Vis. Exp. (2018).

This video describes DNA isolation protocol from stool samples of colon tumor-bearing mice. The extracted DNA is amplified using PCR, and the product is further cleaned using a magnetic beads-based approach. This method can be used to determine the effect of environmental enrichment on colon microbiota and animal mortality.

Protocol

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Genomic DNA Isolation from Stool NOTE: Utilize a commercial kit to isolate microbial DNA from stool following a stool pathogen detection protocol. Remove samples directly from the -80 ˚C freezer and store on dry ice while weighing. Transfer up to 220 mg of stool to a clean mic…

Representative Results

Figure 1: EE and NE housing conditions and stool homogenates (as described in the protocol) Figure 2: 16S Microbiome Library Preparation. (A) Unpurified PCR amplicon products derived from stool genomic DNA.(B) Index…

Materials

1.5 mL Microfuge Tube- RNAse and DNAse free	Any supplier
QIAamp DNA Stool MiniKit	Qiagen	51504	This kit supplies reagents for 50 DNA preparations. Stool Lysis Buffer=ASL; Guanidinium Chloride Lysis Buffer= AL; Wash Buffer 1 with Guanidinium Chloride= AW1; Wash Buffer 2= AW2; Elution Buffer with EDTA=AE
Waterbath (capable of heating to 95)	Any supplier		For 94 degree incubation of stool samples to lyse cells.
Waterbath (capable of heating to 70 degrees)	Any supplier		For 70 degree incubation of stool samples
Ethanol (200 proof)	Sigma Aldrich	E7023
Qubit dsDNA broad Range Assay Kit	ThermoFisher Scientific	Q32850
EB Buffer or 10 mM Tris pH 8.5	Qiagen	19086
Experiment specific primers	Any Supplier
PCR grade water	Any supplier
2X KAPA HiFi HotStart Ready Mix	Kapa Biosystems	KK2601	For Amplicon Amplification (1.25 mL allows 100 rxns).
Agarose for running diagnostic gels	Any supplier
Proteinase K (600 mAU/ml)	Qiagen	19131	Equivalent to 20 mg/ml of proteinase K. Supplied with QiaAmp kit
Agencourt AMPure XP Magnetic Beads	Beckman Coulter	A63880	Magentic beads For PCR cleanup5 mL will clean 250 PCR reactions
Magnetic stand	Life Technologies	AM10027
Fluorometer: Qubit	ThermoFisher Scientific	Q33216
Library Preparation Guide	Illumina		Illumina. 16S Metagenomic Sequencing Library Preparation: Preparing 16S ribosomal RNA Gene Amplicons for the Illumina MiSeq System. https://support.illumina.com/ content/dam/illumina-support/ documents/documentation/ chemistry_documentation/16s/16s/metagenomic-library-prep guide-15044223-b.pdf.
MicroAmp Optical 96-well reaction plate	Applied Biosystems/ThermoFisher
Adhesive clear plate seal	Applied Biosystems /ThermoFisher	4360954	Applied Biosystems/ThermoFisher Microamp adhesive film