Sequencing from chromosomal DNA

Alexander Kraev kraev at bc.biol.ethz.ch
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 bc.biol.ethz.ch
> Lab.of Biochemistry III                            Phone:   0041-1-632-31-47
> Swiss Federal Inst. Of Technology           FAX:      0041-1-632-12-13
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> CH-8092 Zurich                                     /Sasha/kraev.html





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