In article <9407091711.AA14926 at phobos.med.pitt.edu>, bsh at MED.PITT.EDU
(Basavaraju Shankarappa) wrote:
> I have experienced quite a lot of problems specially with respect to
> variability in the DNA yields. The two attempts I made using Qiawell
> resins worked very very beatifully. So we thought this looks like a
> very useful and easy method to prepare DNA and we went ahead bought the
> kit, which is not necessarily cheap. But the next three attempts using
> the same reagents and same batch of plates was a disaster. Some of the
> filtration steps were also very cumbersome and the yield was very low.
> So now, I came back to good old PEG ppt outlined in the DyeTerminator manual.
> The yield and sequence read is not very great but atleast, it gives very
> consitent sequence reads.
> Raj Shankarappa
>bsh at med.pitt.edu>since you mentioned DyeTerminators, i have to assume that you are talking
about using this DNA for sequencing with the ABI 373 Autosequencer. if you
are, i have a couple of suggestions. when we first bought this machine and
i was put in charge of it, i spent a week running the same template DNA
prepared using a number of different methods including: alkaline lysis
alone, alkaling lysis followed by PEG ppt., Qiagen mini and Maxi preps, and
Promega's Wizard Mini and Maxi preps. i used a template which i had gotten
very good manual sequencing results with, and M13 forward and reverse
primers synthesized in-house and EtOH precipitated. i also varied template
amt. as suggested by the tech. rep. from ABI.
Results: generally speaking, if one can get enough DNA from a Qiagen prep
to sequence, then they work beautifully. the problem that i found is that
from one mini-prep we could never get more than 2 reactions worth of DNA to
sequence, and sometimes just one. Alk. Lysis/PEG worked pretty well too
but was a pain in the but. what we decided to go with in our lab as the
standard was a Wizard Mini or Maxi prep (depending on how many primers you
want to use on the template) followed by an EtOH precipitation. it doesn't
take too long (compared to Alk. Lys./PEG) and is much cheaper than the
Qiagen set-up. also, if you use the Wizard mini-preps you don't need to
buy a vacuum manifold or any other toys, just the reagents and some 3ml
syringes. the quality of the Wizard/EtOH preps was pretty close to that of
the Qiagen preps and better than the Alk. Lys./PEG preps.
we have also made the decision to use 500-750 ng of template (vs. the 1 ug
that ABI suggests) to give cleaner sequencing, especially with the Taq
based sequencing systems.
if you have any questions about my results, feel free to e-mail me, or
contact Lily Marr at ABI/Perkin Elmer.
a simple proposal for a more livable usa:
let's ban all guns and personal automobiles...
think about it,
when was the last time you heard of a bike-by stabbing?
just a suggestion
eric c. anderson
anderson at pharmdec.wustl.edu
dept. of molecular biology and pharmacology
washington university school of medicine
st. loser, misery 63110