I re-post previous reply to clumping problem from last month 0.1mg/ml DNase type IIS from Sigma added to the pellet following thawing should solve your problems. Make sure there are some divalent cations in your buffer. The treatment is repeated during processing if clumping reoccurs and we also add DNase to the sort tube (c.02mg/ml) to prevent any problems during sorting - I would avoid extensive vortexing as this may provoke more cell damage. Also strain through 70uM mesh to remove non-DNA clumps (or smaller depending on nozzle size) The best approach is to treat with high concentration as indicated above. This will disperse clumps in a c20s. You can then dilute out DNase activity with buffer. Obviously, don't put EDTA in your buffer or the DNase won't work ! We have no reason to suspect toxicity of DNase under these conditions. Viability & recovery are far better than before we started using it. ################################################################ From: Dr Richard Darley, Ph.D. LRF Differentiation Unit Department of Haematology (A7) University of Wales College of Medicine Heath Park Cardiff, CF4 4XN U.K. tel (+44) 12 22 74 34 85 or (+44) 12 22 74 45 24 Fax (+44) 12 22 74 45 23 e-mail: darley@cf.ac.uk
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