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Sequencing problems. HELP!

Nathan Caffo ncaffo at cais.com
Thu Jul 28 22:18:14 EST 1994

I have encountered several people using automated sequencing (ABI needless
to say) who have similar problems.  Almost without fail if it is a template
problem and they are using Qiagen, they are overloading the columns.  Often
how this happens is someone (and I include myself here) does a prep on a
column that is supposed to yield 20 ug of plasmid.  They are using 5 mls of
culture and get 10ug of DNA.  Convinced that more is better (isn't it
usually), and that the columns should be able to live up to its name they
increase the amount of bugs they put on the column.   The result is usually
exactly what you describe.  Short, noisy sequencing products.  

Hope this helps.  Good Luck
Nathan Caffo
Field Applications Specialist
Applied Biosystems Division

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