Sequencing from chromosomal DNA

Alexander Kraev kraev at
Sat Jun 22 12:55:24 EST 1996

>We have got the sequence of most of our gene, and we would like to
sequence the rest without cloning it. There are only 160 bp missing!!<

1.Can't you just amplify them and sequence the PCR product?
2.Bacterial chromosomes can be sequenced using indirect labelling (aka
Church and Gilbert), but designing such an experiment may be cumbersome. 
3.Theoretically, 1 fmol bacterial DNA (ca.3 ug), a labelled primer with 
Tm of 70 C for cycle sequencing and subsequent PhosphorImager exposure 
should make it. To avoid overloading with template, one should run a 
fairly thick sequencing gel as well (>0.4 mm thick). I hope this helps,

Alexander Kraev, PhD                     Internet:    
kraev at
> Lab.of Biochemistry III                            Phone:   0041-1-632-31-47
> Swiss Federal Inst. Of Technology           FAX:      0041-1-632-12-13
> Universitaetsstr. 16                    Home Page:                                 
> CH-8092 Zurich                                     /Sasha/kraev.html

More information about the Methods mailing list