PCR/sequencing

Steve Marsh marshsr at ccmail.apldbio.com
Tue Jun 3 00:35:49 EST 1997


In article <5mreqg$hva$2 at cronkite.seas.gwu.edu>,
sullivan at gwis2.circ.gwu.edu (Steven Sullivan) wrote:

> Zhonglin Chai (zchai at COBRA.PATH.MONASH.EDU.AU) wrote:
> : Mark Tucker (mtucke39 at IDT.NET) wrote:
> : : does anyone have a one tube PCR/sequencing protocol that works well?  I
> : : appreciate any help given.
> 
> : : mt
> 
> : I have been using Perkin Elmer's ABI PRISM Dye Terminator Cycle Sequencing
> : Ready Reaction Kit wiht AmpliTaq Polymerase, FS. I works beautifully, and
> : very easy to handle (just add your primer and template to a PCR tube and
> : mix with 8ul of the terminator ready reaction mix, 25 cycles, and ethanol
> : precipation, that's it). I routinely use it to sequence both PCR products
> : and plasmid DNA. Email me if you need more details.
> 
> I agree that it works great.  It is, however, quite expensive ($675 for
> 100 rxns).  Also, we've had best success with column-purified reactions
> (Centri-Sep), rather than EtOH-precipitates, and that runs into even more
> money.  Has anyone tried similar kits from other manufacturers? 

When Using our kits for PCR fragments, you can try gel purification after
the initial PCR reaction, before running the sequencing reaction. That
eliminates the expense of the columns. If you buy the kits in larger
quantities you can lower the price. 1000 rxn is $5,500, 5,000 rxn is
$22,500. If you do a standing order the price is further reduced. Before I
joined PE ABD I ran a core facility. With our usage and a standing order
we got the price down to $4.00 per rxn. We used the ethanol precipitation
method to save money. You just need to manually call the sequence around
base 260-270 as there will be a small blob of red color. We very rarely
could not read the sequence under the blob. Hope this helps.
Steve Marsh Field Service Engineer North America
marshsr at ccmail.apldbio.com



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