I am trying to extract bacterial DNA from feces and cecum from mice, to then do PCR. I initially tried with phenol-chloroform and did not succeed. I had better results with CTAB, but the DNA band on the agarose gel is still weak. Would anyone have some protocol with CTAB so I can see what adaptation I could make?
hi Marilia Cavalcanti , i wish this protocol help you:
CTAB protocol
Sample aliquots were spun at 10,000×g to pellet cellular material. After removal of the supernatant, cell pellets were re-suspended in 567 µL of autoclaved and 0.2 filtered TE pH 8 and incubated for 1 hour at 37°C with 30 µL 10% sodium dodecyl sulfate (SDS) and 3 uL 20 mg/mL Proteinase K (Sigma-Aldrich, Castle Hill, NSW, Australia). Samples were then incubated for 10 minutes with 100 uL of 5 M NaCl prepared with sterile water and 80 uL of CTAB/NaCl solution (4.1 g NaCl, 10 g CTAB in 100 mL sterile water). Following incubation, extracts were purified using phenol chloroform extraction, and DNA was recovered by isopropanol precipitation. Pelleted DNA was washed twice with cold 70% ethanol, allowed to air dry, and re-suspended in 50 µL of sterile water.
you can try potassium acetate method of DNA extraction.
some Extraction kits are also online available eg. himedia DNA extraction kit.
best wishes.
Utsa Roy, Ezzouhra Elmaaiden and Dalveer Singh Somal thank very much for the answers!
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