Q: Direct PCR prod. sequencing

Eric C. Anderson anderson at pharmdec.wustl.edu
Thu Mar 16 14:23:34 EST 1995


i have been using the USB Exonuclease I/Shrimp Alk. Phos. procedure for
treating  pcr reactions for automated sequencing using the ABI 373
sequencer (FWIW i have no interest in either company, just use the
products) for quite some time now with good results.

i just spoke to someone in the dept. though who said that she was told (not
by me) that she only needs to use 10ng of ExoI/sAP treated PCR product in
her sequencing reaction, not the 750-1000ng that ABI says to use for a
plasmid.  now, i understand that it might not be necessary to use quite so
much as that (1/4-1/2 of that amount would probably suffice), but 1/75 of
that amount?  is that really going to work.

i have been assaying DNA conc. by taking A260 readings.  i know that there
is probably some interference by the enzymes and leftover buffers, but
using those readings to convert to ug/ml and then using "750ng" of that
product has worked very well.  could i be mis-measuring the concentration
by a factor of 75?

also, this same person said that some of her reactions didn't work using
750ng so she's going to try using 10ng.  does this make sense to anyone? 
it certainly doesn't to me.

thanks for any insight that anyone may be able to provide,

eric

-- 
eric c. anderson                                
anderson at pharmdec.wustl.edu
dept. of molecular bio. and pharm.               (314)362-3963 (lab)
washington univ. school of medicine              (314)862-2435 (home)
660 s. euclid box 8103                           (314)362-7058 (FAX)
st. louis, mo 63110



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