Coordinated regulation of gene expression by E ... - Jacobs University

MATERIALS AND METHODS
2.2 Methods
2.2.1 Agarose gel electrophoresis
Electrophoretic separation of DNA was carried out using 0.8-1.5% (w/v) agarose gels
prepared in 1X Tris-borate (TBE) or Tris-acetate buffer (TAE). The gel buffer and
running buffer are identical. For all applications (except for topology gels) ethidium
bromide (0.3 μg/ml) was mixed with the gel before casting. Various size of gel
chambers (8 X 10 to 19.5 X 19.5 cm) were used to cast the gel and the DNA to be
separated was mixed with 1/6 th the volume of loading dye, with size markers (λ pst, see
below) when necessary. Separation was carried out at 0.5 V/cm for topology gels and at
1-5 V/cm for other purposes. Subsequently gels were observed under UV light in gel
documentation equipment (Biodoc analyze from Biometra ® ) and photographed when
necessary.
DNA ladder for size marker was obtained by digesting overnight 25 μg of λ
DNA (New England Biolabs) with PstI restriction enzyme in an appropriate buffer at
37 o C.
2.2.2 Small and medium scale isolation of plasmid DNA using
Qiagen kit
Small and medium scale plasmid isolation was performed using QIAprep Spin
Miniprep Kit and QIAGEN Plasmid Maxi Kit respectively by following the
manufacturer’s instruction. The E.coli cultured in 2X YT and LB has been used for
extracting plasmid.
24