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

MATERIALS AND METHODS
measure of opening of the two strands of DNA during processes such as initiation of
transcription.
All the components (except KMnO4) are mixed on ice and equilibrated at
37oC for 5 minutes before adding 2 μl of 100 mM KMnO4. After exactly 30 seconds,
the footprint is stopped by the addition of 2 μl of β-mercaptoethanol (14M). To this 100
μl of TE (pH 8.0) is added and then the DNA is extracted with phenol: chloroform and
precipitated as described above. The pellet of footprinted DNA is dissolved in 15 μl of
water and is subjected to primer extension with labeled primer using Taq polymerase
and 1 mM final concentration of dNTP mix for six cycles in a PCR machine (Peltier
Thermal Cycler PTC 100 & PTC 200 from MJ research). The product is resolved on
a 6% polyacrylamide gel along with a sequencing reaction and visualized by
phosphorimaging. The di-deoxynucleotide termination sequencing reaction of the
plasmid DNA and the primer used for footprinting and PCR was performed using
commercially available sequencing kit from USB (Sequenase Version 2.0 DNA
Sequencing Kit) following the manufacturers instruction.
2.2.19 β-Galactosidase assay
Beta-galactosidase assay was performed following the protocol of Sadler & Novick
[Sadler & Novick, 1965]. The β-Galactosidase assay was performed on freshly diluted
(1:30) overnight cultures after one hour of dilution cultures in exponential phase. β-
Galactosidase activity is calculated as a function of, colorless ONPG substrate turning
to yellow nitro phenol product (measured at OD420) and the time it takes to turn yellow
by using the formula:
β-Galactosidase activity = OD420 X 8.5 X 100
OD600 X time (minutes)
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