I am working on virulence factors and proteins associated with H. pylori infection. For this, I have collected gastric biopsies and treated them with T-PER Tissue Protein Extraction Reagent ( 25mM bicine, 150mM sodium chloride; pH 7.6 ) to extract total proteins according to manufacturer instructions (mentioned below) for MS analysis.
1. Weigh biopsy samples.
2. Used 100 μL of a reagent to extract concentrated protein and homogenize.
3. Centrifuged the sample at 10,000 × g for 5 minutes to biopsy debris.
5. Collected the supernatant and continue with purification and MS analysis.
Due to the limitations of getting more biopsies from patients, I wanted to extract DNA (for virulence factors) from tissue debris after centrifugation (of step 3) if possible.
Could you people please guide me?
I'd just add DNA extraction reagents and continue, the remains of the bicine and the NaCl shouldn't interfere. Depending on the storage of the samples, and the extent of homogenization (shearing forces!) DNA might have degraded to some extent, but you'll see that on a gel and if you PCR for short amplicons, it shouldn't be a big problem for an exploring experiment.
I imagine that your bicine and protein extraction won't damage your DNA from the biopsies. If your homogenization still leaves solid pieces that are in the pellet, they should be protected by shearing forces. Most DNA extractions start with a Proteinase K treatment anyway so doing a protein purification shouldn't hurt. We've also found that you may have to play around with your lysis buffer components to get at the pathogen genetic material in case you want to assay for the quantity of H.pylori in your biopsies. Our bacteria, T.pallidum needed SDS addition to get it away from the tissue and EDTA to stabilize the DNA.
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