ALF Express Problems
rpa.groot.oncol at med.vu.nl
Tue Aug 29 02:40:11 EST 1995
In article <41ncoh$i10 at usenetz1.news.prodigy.com>, RLZP39B at prodigy.com (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.
> DOUGLAS BUECHTER RLZP39B at prodigy.com
In our laboratory internal labeling with fluorescein-15dATP is a
well know problem.
According to Wiemann et.al.,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)
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