Protocol for Cosmid Cycle sequencing?

colossus... s535290 at aix1.uottawa.ca
Wed Jun 18 12:10:24 EST 1997


Armin, 

I think you will have to determine optimal conditions on your own, since
the conditions in the ABI manual are only guidelines. I would definitely
say that you should keep primer concentrations similar to those mentioned
in the manual, but definitely use more DNA than you would use if you were
sequencing a plasmid. I even notice that sequencing a "larger" plasmid
requires me to use a little bit more DNA than when sequencing a smaller
plasmid (ie. an 8Kb insert in pBluescript vs. a 1.5 Kb insert in
pBluescript). Since cosmids are so large, you definitely want to use more
DNA. But you should try and determine standard conditions based on the
cosmids you use, as well as the purification technique that you are using.
Once you standardize this, you can use the same conditions over and over.

Good luck,

Ed

ps. I do know someone who does automated sequencing of cosmids and can ask
him the amounts of template and primer used - if you need me to find this
out for you drop me a line

> Does anybody know of a reliable protocol for sequencing cosmids by cycle
> sequencing? Of special interest for me are the amounts of DNA template
> and primer to be used in the cycling reaction. I tried the sequencing
> reaction with the standard template amounts for plasmid DNA (about 500
> ng which yielded good results in our standard plasmid sequencing
> reactions) and with significant higher template concentration (about 2
> µg) but got no usable sequence using standard sequencing protocols
> referring to the ABI 373/PE ABI Prism Cycle Sequencing manual. 
> 
> Every help is greatly appreciated.
> 
> Thank you in advance,
> Armin Michel
> 
> 




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