I have samples that contain bacteria stored in glycerol. These samples are about 9-10 years old and have been in the freezer since then. The sample volume is pretty small (50 - 100 ul). The bacterial cells are very likely dead by now, so they can't be cultured. What would you recommend for bacterial DNA extraction in order to get rid of the glycerol which would inhibit PCR? Thanks!
I would resuspend this volume in TE or Ringers buffer before centrifuge at maximal speed for 5-10mins. Then remove supernatant, the pellet I would recommend a 2min bead beat (5000xrpm) with 500ul of lysis buffer (please see Yu and Morrison 2004 ://www.ncbi.nlm.nih.gov/pubmed). Following the described extraction in the paper will produce good quality DNA for pcr or sequencing.
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