Taq pcr master mix

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:

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.

The thermal cycler program was adjusted & preceded as shown in 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)].

Twenty-five soil samples were collected from healthy banana rhizosphere soil in an area affected with Fusarium wilt of banana in Zhanjiang City, Guangdong Province in November of 2015. From a healthy plantain garden where bananas has been grown for more than 10 years, rhizosphere soil was collected at 10–30 cm of the root of healthy plants. Soil samples were collected and placed inside a sterile plastic bag, sealed, and preserved in an ice box. In the laboratory, the roots and stones were removed and stored at 4 °C before the actinomycetes were isolated.
Pathogenic fungi strains includes Fusarium oxysporum f. sp. cubense Race1 (FOC.1), F. oxysporum f. sp. cubense Race 4 (FOC.4), Curvulatia fallaxis (CFO), Colletotrichum gloeosporides (CG), and Alternaria tenuissima Maa (MAA), Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), and Staphylococcus aureus (S. aureus)were provided by the Institute of Environment and Plant Protection. Taq PCR Master Mix and bacterial genomic DNA rapid extraction kit were purchased from Sigma-Aldrich Co. (USA).
“Gause’s no. 1 culture”: 20.0 g Soluble starch, 0.5 g NaCl, 1.0 g KNO₃, 0.5 G K₂HPO ₄·3H₂O, 0. 5 g MgSO ₄·7H₂O, 0.01 g FeSO ₄·7H₂O, 20 g agar, 1000 mL H₂O