ALF Express Problems

RPA Groot rpa.groot.oncol at
Tue Aug 29 02:40:11 EST 1995

In article <41ncoh$i10 at>, RLZP39B at (Douglas Buechter) says:
>We recently purchased an ALF Express DNA sequencing system from Pharmacia.
>  The apparatus works well but we are having a lot of problems getting 
>Pharmacia's sequencing kit to work when we use internal labeling with Cy5-
>dATP.  Labeled primers do ok but with the internal labeling we get either 
>no or litte signal in trying to sequence plasmids that sequence ok either 
>by manual Sequenase or with Cy5- labeled primers.  We typically do Wizard 
>Minipreps but have also tried a couple of times to sequence Qiagen-
>prepped plasmids with no better sucess.
>   Thanks for any suggestions.

In our laboratory internal labeling with fluorescein-15dATP is a
well know problem. 

According to Wiemann,Biotechniques 1995 Vol.18 No.4 p688-697, 
this is a primer problem; an 'A*' must be as close at the primer binding 
site as possible (not more then 2 nucleotides downstream).
When an 'A*' is incorporated directly after a primer,the sequencing 
reaction should be succesfull.  

So 25-50% of all primers should work.(wich I haven't checked for our primers)

Richard Groot

More information about the Methods mailing list