Double stranded DNA sequencing
Mon Feb 14 00:51:54 EST 1994
In article <1994Feb11.212054.4104 at galileo.cc.rochester.edu> PHIP at BPHVAX.BIOPHYSICS.ROCHESTER.EDU (RICHARD P. PHIPPS) writes:
>I am currently attempting to sequence double stranded plasmid DNA from
>minipreps using the USB sequenase 2.0 Kit. Unfortunatly after trying several
>DNA isolation methods that I could think of or find I am not having good
>results in that > 70% of my reactions produce either no bands or bands in all
>four lanes all the way up the gel. I am interested in a miniprep DNA isolation
>technique (Kit or otherwise) that reliably yields 50% or greater readable
I have used a modified mrthod of the normal alkaline-lysis
method, usually with good sucess.
carry your miniprep to the cleared lysate stage
extract with ~1/3 volume of phenol that has been washed x3
with 50mM Na Acetate(pH4.0), vortex, leave on ice for 5 min.
Spin in microfuge x 5 min to separate the phases. Remove the
upper aqueous layer (NB avoid any of the interface, take
less sup if necessary). Extract with 1/3 vol of 24:1 CHCl3:
isoamly alcohol. Spin. Remove upper aquous layer.
Precipitate DNA and wash as normal(we pptte with isopropanol
at 0.6 vol)
I hope this works for you
Uni of Sydney
mts at biochem.su.oz.au
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