The Vitek-2-confirmed ethanol-resistant K. pneumoniae and E. coli isolates were tested for the Adh gene by PCR. Briefly, bacterial chromosomal DNA was extracted according to the method described by Wilson [50 (link)] to get the DNA templates. Adh gene amplification by PCR was conducted using a pair of primers (Sigma) selected according to [9 (link),51 (link)] & the sequence of the primer used was:
The PCR reaction was conducted in a final volume of 25 μL which contained:Table 1 [52 (link)]:
Agarose gel (1.5%) electrophoresis of the amplified Adh gene was conducted using the DNA molecular marker (100 bp DNA Ladder; Lonza Inc., Rockland, MA, USA) to detect the expected (1038 bp) bands visualised by staining with ethidium bromide (EB) [53 (link)].
The PCR reaction was conducted in a final volume of 25 μL which contained:
A volume of 12.5 μL of Taq PCR Master Mix after being briefly vortexed to avoid localised differences in salt concentration.
The primer solutions were thawed on ice & mixed well before use. One μL of each primer was added to the PCR tube.
A volume of 5 μL of template-extracted DNA was added to each tube.
A volume of 5.5 μL of nuclease-free double distilled water was added.
Agarose gel (1.5%) electrophoresis of the amplified Adh gene was conducted using the DNA molecular marker (100 bp DNA Ladder; Lonza Inc., Rockland, MA, USA) to detect the expected (1038 bp) bands visualised by staining with ethidium bromide (EB) [53 (link)].
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