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

MATERIALS AND METHODS
2.2.3 Restriction Digestion
Digestions of DNA with restriction enzymes were performed according to
manufacturer’s instructions using recommended buffer systems and at the appropriate
reaction temperatures. Generally, 2-10 units of enzymes were used per μg of DNA.
Plasmid DNA was usually digested for 1-3 hours and the completion of the reaction
monitored by gel electrophoresis.
2.2.4 Polymerase chain reaction
PCR was primarily employed to extend the labeled primer (2 pmol) from a footprinted
DNA template (around 1 μg). In a reaction volume of 20 μl, using Taq polymerase and
1mM final concentration of dNTP mix, the reaction was performed for 6-9 cycles. The
reaction conditions were as follows: Denaturation at 94 o C for 30 seconds, annealing at
52 o C for 1 minute, elongation at 72 o C for 1 minute.
2.2.5 Preparation of competent E.coli cells using calcium chloride
Calcium chloride competent cells used for transforming plasmids into E.coli were
prepared following the protocol described by Sambrook et al. [Sambrook and Russell
2001]. The competent cells prepared was stored at -80 o C in aliquots of 100 μl.
2.2.6 Transformation of E.coli cells
Transformation of plasmids into E.coli was performed as described by Sambrook et al.
[Sambrook and Russell 2001] using calcium chloride competent cells. The standard
protocol was followed except for different heat shock temperature. Heat shock was
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