ABI 373 DNA sequencer

Steve Marsh marshs at cco.caltech.edu
Fri Nov 11 14:09:22 EST 1994


In article <39qt17$oef at mserv1.dl.ac.uk>, "Jacqueline Vanderstappen"
<jacqueline.vanderstap at agr.kuleuven.ac.be> wrote:
> 
>> We've recently bought the upgrade version (34cm WTR). Sometimes we have 
> problems with directly sequencing dsPCR products with the Taq dye terminator 
> kit. It's not a problem of reactions because the sequencing with the same 
> reactions have worked with the previous version of ABI 373 (24cm WTR). 
> We tried to use the Sequenase dye terminator kit for dsPCR products but it 
> didn't work? Is there somebody who has good experience with it? We perform 
> the sequencing directly on double-stranded PCR products.

> Vander Stappen Jacqueline
> KULeuven
> Laboratory of Gene Technology
> W.deCroylaan 42
> 3001 Heverlee
> Belgium
> e-mail: Jacqueline.Vanderstap at agr.kuleuven.ac.be 

I have Been sequencing PCR products on the 373 Stretch. One thing to check
is the relative concentration of the pcr products as compared to a plasmid.
We found that we needed to dilute our PCR products 1:10 to get the
concentration right. The PCR product was ~500 bp long. Most of the plasmids
we are doing are ~5kb. Other than that we doing the reactions following the
protocol. I have done both Dye primer and Dye terminator reactions, but not
sequenase. The one thing that is critical is cleaning up the PCR products
before sequencing them. We gel purify them before we sequense. Hope this
Helps.

Steve Marsh.
DNA Sequencing Core Facility at CalTech.



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