cycle seq. of lambda

Bruce Roe broe at aardvark.ucs.uoknor.edu
Sat Oct 3 08:04:00 EST 1992


In article <1992Oct2.132129.1956 at gserv1.dl.ac.uk>, gessler at convex.HRZ.Uni-Marburg.DE (Gessler Manfred Dr.) writes...
>We would like to sequence lambda clones with cycle sequencing on an ABI
>sequencer. Has anyone done this with dye-terminators, or do we need labelled
>primers to get useful sequences? 
>Any tips on protocols for DNA prep, oligo length etc. are welcome!
> 
>Thanks in advance
> 
>Manfred Gessler
>Uni Marburg
> 
>gessler at convex.hrz.uni-marburg.de
>FAX (49)6421-285630

Manfred,
	We have tried this with labeled primers with mixed success.
The main problem was that template concentration needed to be optimized
and getting beautiful, intact template can be a chore.  Doug Berg with
the group at BRL and Palazzolo at LLB have done some studies. 

Try looking at:

 Phadnis, S.H., Huang, H.V. and Berg, D.E., Proc. Natl. Acad. Sci. USA 86,
 5908-5912 (1989).

 Strathmann, M., Hamilton, B.A., Mayeda, C.A., Simon, M.I., Meyerowitz,
 E.M.and Palazzolo, M.J., Proc. Natl. Acad. Sci. USA 88, 1247-1250 (1991).

for some ideas and suggestions (even these papers used radio-labeling).

Cheers.....bruce
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 \  Bruce A. Roe                     Dept. Chemistry and Biochemistry /
 /  BROE at aardvark.ucs.uoknor.edu     University of Oklahoma           \
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