Freeze Shearing of DNA

Steve Griffiths sgriffit at rpc.unb.ca
Fri Aug 2 12:39:38 EST 1996


  Our group has recently been looking at ways to rapidly evaluate increases in 
bacterial load using PCR amplification of a universal 16s ribosomal 
sequence.The system works beautifully as a means for comparing influent 
seawater with that in tanks containing fish larvae with one problem---We were 
using a  prep of ultrapure Ecoli DNA figuring that if 5 femto grams of DNA 
represents a single bacterium, we could translate  amplified product band 
intensity into the number of "Ecoli units" per mL of water.While the linear 
range of band intensity over 2 fold dilutions between 10E6 and 100 was 
exceptional immediately after preparing the Ecoli standards,when the aliquots 
were thawed the sensitvity appeared to decrease over time.For example if an 
aliquot was thawed after 1 week of storage, sensitivity (ie appearance of 
amplified product) would drop by 2 2-fold dilutions. After a month of storage 
it appears to have dropped another two dilutions.
        The question is this(phew!).Even though the 50ng aliquots are 
continually stored at -20degC in polystyrene racks,  would you expect the 
bacterial genomic DNA to degrade or shear yto the extent that I'm suggesting?
 Any communication would be appreciated. STEVEg



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