Quantitation of small amts of DNA

Nicola Fidler bt048478 at dingo.cc.uq.oz.au
Wed Aug 10 21:54:12 EST 1994


hardy at mighty.fccc.edu (Richard R. Hardy) writes:

>We're starting to do lots of taq cycle sequencing using ABI kits on a 373A
>automated sequencer and have noticed that we need to pay close attention
>to the amount of plasmid template if we want to get "the best" data (long
>reads free of Ns). Previously with manual dideoxy sequencing we estimated
>DNA amounts by ethidium staining, but this may not be good enough for
>optimal automated cycle sequencing (ABI rep said, use 1 ug of template;
>1.3 is too much, 0.8 is probably still OK; that's a pretty narrow
>window).  
 [stuff deleted]

Our group had similar problems but now now problems - we use the whole 
product of a 1.5 ml miniprep and routinely read at least 500 bp.
Good luck.
Nick



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