Dear Dr. Whistler,
Regarding your questions about methods for sequencing PCR products
on the ALFexpress, using internal labelling with CY5-dATP, I checked into
the situation. Pharmacia Biotech does produce a kit for preparing ssDNA
templates from PCR products, called "PCR Template Prep for ssDNA Sequencing"
catalog number 27-9200-01. The kit uses Lambda Exonuclease to selectively
digest the strand of DNA which is 5' phosphorylated. Using this kit, it is
easy to prepare specific strands of DNA, by regulating which of the PCR
primers is phosphorylated. If you have further questions, please feel free
to contact your local Pharmacia Biotech representative.
Sincerely,
Marc A. Goldstein, Ph.D.
Applications Specialist
Pharmacia Biotech, Inc.
At 03:06 PM 3/13/96 GMT+0200, Dr. Toni Whistler wrote:
>>We have just recently purchased the ALF-express and I am a little at
>sea as to getting things going. The machine is running well using
>cy5-labelled primers and sequencing off plasmid. The majority of the
>sequencing done in this department is off PCR products, using the
>incorporation of cy5-dATP.
>My main complaint so far is the very poor literature that goes with
>this machine .... protocols are scarce.
>I have several questions.
>1) Sequencing PCR product, I need to make it ss-ed. Asymmetric PCR or
>ExoI digestion.
>2) Clean up of ssDNA, what is the best method.
>3) What sort of concentrations of template (450bp to some of 1600bp)
>is used, what are the primer concentrations.
>>I would appreciate any and all advise. If anyone has protocols they
>use in their lab, these would be most appreciated.
>Thanks
>Toni
>>>>**********************************************************
>> Toni Whistler, PhD
> Department of Molecular Virology
> National Institute for Virology
> P Bag X4
> Sandringham, 2131
> Johannesburg
> South Africa
>>E-mail: Toniw at niv.ac.za>Tel: (011) 882 9910
>Fax: (011) 882 0596
>>**********************************************************
>>
\\/\\/\\/\\/\\/\\/\\/\\/
Marc A. Goldstein, Ph.D.
Applications Specialist
Pharmacia Biotech, Inc.
1-800-526-3593, 1, 5057
magoldst at ix.netcom.com
