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

MATERIALS AND METHODS
given using a heating block (Eppendorf® Thermomixer Compact T1442) at 37 o C for
LZ strains and at 42 o C for CSH50 strains.
2.2.7 Purification of nucleic acids by Phenol: Chloroform
extraction
Purification of nucleic acids by phenol: chloroform was performed as described in
Sambrook et al [Sambrook and Russell 2001].
2.2.8 Concentrating and quantification of nucleic acids
The DNA and RNA were concentrated by ethanol precipitation method as described by
Sambrook et al., [Sambrook and Russell 2001] differing from the standard protocol in
the salts used and the final drying step. When concentrating DNA the salt solution used
was 1/10 th volume of sodium acetate (pH 5.2) and in the case of RNA 1/5 th the volume
of ammonium acetate was used. Finally the pellet of nucleic acid was either air dried by
keeping the tube open at room temperature or dried using a speedvac (Eppendorf ®
Vacufuge concentrator 5301). Quantification of the nucleic acids was done using
Nanodrop (ND-1000 Spectrophotometer).
2.2.9 Polyacrylamide sequencing gels
The preparation and resolving, of DNA probes and sequencing reaction products, was
performed as described by Sambrook et al., using 6% polyacrylamide gels (composition
for 500 ml: 210 gm urea, 50 ml 5X TBE, 74 ml Rotiphorex 40 (Carl Roth GmbH).
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