A modified, abbreviated version of the Maxam-Gilbert sequencing is described. A complete description including the methods to prepare and isolate asymmetrically labeled fragments of DNA and to prepare and run sequencing gels can be found in Methods in Enzymology 65: 497-559.

Reagents:

-Dimethylsulfate (DMS) Extremely dangerous.

-Hydrazine (HZ) Very dangerous.

-Formic acid

-Piperidine. Stock is 10 M. Dilute to 1.0 M just before use.

-100% Ethanol

-dH2O

-Acetic Acid. Glacial is 17.4 M. Dilute to 1 M.

-0.3 M Sodium acetate (pH 5.2)

-5 M NaCl

-10 N NaOH

-500 mM EDTA

-tRNA. Stock solution is 1mg/ml in dH2O.

-Calf thymus DNA. Stock solution is 1 mg/ml in TE.

Buffers:

DMS buffer

50 mM sodium cacodylate (pH 8.0)

1 mM EDTA

DMS stop

1.5 M sodium acetate (pH 7.0)

1.0 M mercaptoethanol

100 μg/ml tRNA

HZ Stop

0.3 M sodium acetate

0.1 M EDTA

25 μg/ml tRNA

Loading buffer

80% (v/v) formamide

50 mM Tris-borate (pH 8.3)

1 mM EDTA

0.1% (w/v) xylene cyanol

0.1% (w/v) bromophenol blue.