Genomic DNA Extraction from Tumor Specimens

Margaret A. Keller, Ph.D. keller at email.chop.edu
Wed Jun 5 13:17:45 EST 1996


We are attempting to isolate DNA from tumors using an Applied Biosystems
Nucleic Acid Extractor Model 341. The homogenized sample (liquid nitrogen
frozen, then broken up with a mallet) can be added to the instrument in
lysis buffer and the machine performs organic extraction and
precipitation. 
Initially, when the phenol was added to the lysate, there was a lot of
foaming, which we interpreted to mean that cells remaining unlysed in the
lysate buffer were lysing at the organic step. We altered our protocol to
include an overnight digestion with proteinase K at 65C. Now we either get
very small yields, or there is poor phase separation during the
extraction, leading to sample loss. Does anyone have any suggestions about
how to treat the samples prior to loading on the machine? It would also be
interesting to hear about other instrumentation that can extract genomic
DNA from tissues. We are interested in keeping this methodology
semi-automated for high throughput. I should mention that the machine
works fine with lymphocyte or bone marrow samples.  
Please reply to napcore at email.chop.edu.
Margaret Keller



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