Stool Microbial DNA Isolation: Studying the Effect of Environmental Enrichment on Microbiome Diversity in Murine Tumor Model
Published: April 30, 2023
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…
Offenlegungen
The authors have nothing to disclose.
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 |
Diesen Artikel zitieren
Stool Microbial DNA Isolation: Studying the Effect of Environmental Enrichment on Microbiome Diversity in Murine Tumor Model. J. Vis. Exp. (Pending Publication), e20332, doi: (2023).