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…
Disclosures
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
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).