First of all, the reason that you need to have the DNA clean is because of
the loading method onto the capillaries. Loading is done electrokinetically
(running a current). Any charged molecules in your DNA prep are going to
compete with the DNA in being loaded into the capillary. Therefore,
proteins and salts will interfere and the result will be poor signal and
poor sequence data.
The preps we have tried are Promega's Wizard and Wizard SV, and Qiagen (all
types). We have found that these all give good sequence, but find that
Qiagen and Wizard SV are slightly better. Note, you can clean up the old
Wizard with EtOH precipitation and then it works fine.
We have tried Centri-Cep (Princeton Separations) and Millipore PCR-cleanup
columns (after the seq. PCR step) and have found that the Centri-Cep give
cleaner results. We have yet to try the Edge Biosystems 96well cleanup, but
will try next month. With the success of these columns, we have moved to a
96-well system using a Savant Speed-vac to dry down samples, and a bench-top
centrifuge that holds plates. We are looking into a PCR machine that has 4
places for 96-well plates. If you have any suggestions, please let me know.
We have been using the same amounts of DNA that we used in our 373's or
about 250ng/reaction. And 10ng of primer. We started with 1/2 reactions
and when we get settled we are going to try smaller reactions. Note, most
of the DNA submitted is DS. We have seen PCR products work just fine with
Qiagen clean-up.
Hope that helps, feel free to ask more questions.
Dina Leviten
ICOS, Corp.
Bothell, WA
> -----Original Message-----
> From: Paul Morrison [SMTP:p_morrison@dfci.harvard.edu]
> Sent: Friday, May 14, 1999 9:46 AM
> To: Recipients of ABRF List
> Subject: RE: DNASeq: New 3700?
>
> Reply to: RE: DNASeq: New 3700?
> To say the least this is the most encouraging news I've heard on the 3700
> read length. Instead of everyone calling Dina up, (I tried and it's now
> always busy), let's convince her to extrapolate on this tantalizing bit of
> info.
> How is the template quantified, desalted? What is the template prep? How
> clean is clean? Have you burnt out a 96 array yet? Are they all working?
> Stuff like that.
> -Paul
>
> Paul Morrison
> Molecular Biology Core Facilities
> Dana-Farber Cancer Institute
> Jimmy Fund Two
> 44 Binney Street
> Boston, MA 02115
>
> paul_morrison@dfci.harvard.edu
> http://mbcf.dfci.harvard.edu
>
> 617-632-3082
> fax 632-4814
>
>
>
> Leviten, Dina wrote:
> >Lisa,
> >
> >We just got our 3700 installed last week and here's a breif synopsis of
> how
> >things are going. Bottom line, machine is great--software needs help.
> >
> >The install went well and the instrument works great. For long reads, it
> >takes 4.5 hrs (of that 2.5hrs are collection of data). Using the machine
> is
> >easy and is sure beats pouring and loading gels. The software right now
> is
> >another story! It is pretty intuitive, but in these early stages, there
> are
> >tools that need to be added to make our lives easier. Most of the big
> >problems we have should be easily fixed with some software changes. The
> >biggest problems are sample sheet preparation and printing. At this
> point
> >it is tedious, but we have come up with a couple work-arounds that have
> cut
> >down on the time. We are getting a database system that should address
> most
> >of the sample sheet and printing problems.
> >
> >Data looks good. In the run time we get about 700 bases. Clean DNA is
> key.
> >
> >Data files are in "dos" but can be converted to "abi1" with a program
> called
> >"SetItsType"ver1.3 (found free on the internet). This enables programs
> like
> >Sequencher to see the chromatograms.
> >
> >Overall, we are really happy with our purchase and everyone loves NO
> >BACKLOG!!! We are exclusively using the 3700, where we were using 2
> stretch
> >373's. The 3700 can run 5 96-well plates in a 24hr period, where you run
> >one during the day and can set up the other 4 for an overnight run.
> >
> >If you want the nitty-gritty details, I can supply them, but by the time
> you
> >would get an instrument, these problems will probably be gone (I'm
> >optimistic!)
> >
> >Hope this helps!
> >Sincerely,
> >Dina Leviten
> >ICOS, Corp.
> >Bothell, WA
> >
> >
> >> ----------
> >> From: lisa bibbs
> >> Sent: Thursday, May 13, 1999 5:03 PM
> >> To: Recipients of ABRF List
> >> Subject: DNASeq: New 3700?
> >> >> Does anyone have one and would like to comment? We have reached are
> >> capacity and are trying to decide about next years budget.
> >> >> Thanks in advance
> >> Lisa
> >> >> >
> >RFC822 header
> >-----------------------------------
> >
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> > From: "Leviten, Dina" <dleviten@icos.com>
> > Old-To: Recipients of ABRF List <abrf@aecom.yu.edu>,
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> > <bibbs@scripps.edu>
> > Subject: RE: DNASeq: New 3700?
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