Process it for what? Histology, protein analysis, gene analysis?
Dear Soto,gene and protein analysis, i mean any method which needs cell rupture
we used TRITON X-100 for the perforation of brain tumor cell membranes. take 1ml of the 0,2% solution for 20 minutes (into a 2ml well). we were able to culture intracellular bacteria (listeria monocytogenes) after this procedure.
Are you worried about personal safety ?
1. ALL human samples must be handled in BSLII facility (a BSLII cabinet or hood is a good start).
2. All solutions, pipettes plastics etc. must be treated with 1% fresh chlorox for 10 misn and then autoclaved. Alternatley, if you can collect the waste safely in biohazard bags, autoclaving may be enough.
3. If you know there is a specific LIVE virus or bacteria, the level of safety may be have to be increased depending on the tuype of bacteria/virus. This is absolutely no trivial matter. Look up NIH site to start with,.
The personal safety is an important issue. Nonetheless, returning to the protein and nucleic acid analysis, do you want brain tissue proteins and nucleic acids, or are you interested in being able to analyse both, the pathogen and the tissue?
Dear soto, i want to isolate DNA or protein without bacteria and virus etc.
Hi, this might not be important anymore, but to get cell DNA/protein from virus infected cells I'd freeze and thaw the sample to break open the cells, followed by sonication for 30-60s on ice.
The virus will survive that (unless you have some very unstable virus) and you can try to do a protein/DNA isolation from that. If not, maybe try to follow a standard virus isolation protocol but discard the virus instead of the cell pellet.
Alternatively you could just try to do a general protein/DNA purification protocol. Viral protein will be fairly rare compared to all the cellular protein, and with DNA your PCR should only amplify your target DNA.
hope that helps
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