Chloroform isoamyl alcohol

To extract genomic DNA for use in spoligotyping, the frozen regrown MDR-TB isolates were defrosted on a thermomixer at 60°C. 70 μL of 10% SDS (Sigma, USA) and 50 μL of 10 mg/mL proteinase K (Sigma, USA) were added to the thawed sample and incubated for 1 hour at 60°C at 400 rpm on a thermomixer. 100 μL of 5 M NaCl and cetyltrimethylammonium bromide (CTAB) (preheated to 60°C in water bath) was added and mixed by inversion before further incubation for 1 hour at 60°C and 400 rpm. The mix was then incubated at −70°C for 15 minutes before being allowed to thaw and reincubated for 15 minutes at 60°C and 400 rpm. 700 μL of chloroform/isoamyl alcohol (Sigma, USA) (24 : 1) was added and the mixture was centrifuged for 10 mins at 13,000 rpm. The resulting upper aqueous phase was transferred into a clean tube containing 700 μL of cold isopropanol (Sigma, USA) and incubated at 4°C overnight. The next day the solution was centrifuged for 10 minutes at 13,000 rpm and the supernatant was discarded. The tube was washed with 100 μL of 80% ethanol (Sigma, USA) by centrifuging for 10 minutes at 13,000 rpm. The supernatant was discarded and the pellet DNA pellet was air-dried before being resuspended in 50 μL of DNase-free water and kept at −40°C.

Tris hydrochloride (pH 9.0), sodium dodecyl sulfate (SDS), lithium chloride (LiCl), ethylenediaminetetraacetic acid (EDTA), phenol (pH 8.0), chloroform–isoamyl alcohol (24:1; v/v), phenol–chloroform–isoamyl alcohol (25:24:1; v/v/v), sodium acetate (3 M, pH 5.2), sodium chloride (NaCl), polyethylene glycol 8,000, and pepsin were obtained from Sigma‐Aldrich (St. Louis, MO). RNA oligos with 3′ end 2′‐O‐methylation were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA).

Bacterial pellets were thawed on ice. Cells were first resuspended and incubated in 1 mg/ml of lysozyme (Sigma) prepared in T.E. buffer and then completely lysed by the addition of 1 volume cell lysis buffer (Puregene). Spike-in RNAs were introduced at this stage.
The samples were extracted with acidic phenol/chloroform (Sigma) 2–4 times and then once with chloroform:isoamyl alcohol (24:1, Sigma). The RNA was then precipitated with the addition of 1/10 volume 5 M NaCl and 1 volume isopropanol on ice or in −20°C. RNA was pelleted and washed twice with 70% ethanol, resuspended to 100–200 ng/μl in nuclease-free water (Ambion or Cellgro), then digested with DNaseI (NEB) to remove contaminating DNA. The RNA was then purified as described. After resuspending in water, the RNA was used immediately.

The procedure for extracting DNA was based on a modified protocol described by Douterelo et al.^{12 (link)}. Filters with concentrated biofilm solution, were placed into a 2 mL Eppendorf tubes. Then 740 μl of SET lysis buffer (40 mM EDTA, 50 mM Tris–HCl, pH 9, 0.75 M sucrose) was added and the filters were crushed with sterilized pestles (autoclave 20 min 121 °C). Ninety microlitres of lysozyme (9 mg/ml) was added and filters incubated at 37 °C for 30 min with shaking (100 rpm) in a Hybaid hybridisation oven (Thermo Scientific, UK). Subsequently, 90 μl of sodium dodecyl sulphate (SDS) and 25 μl of proteinase K (20 mg/ml) were added to the tube and the sample incubated at 55 °C for 2 h with shaking in a Hybaid oven. The supernatant (aqueous phase) was transferred to a clean 2 mL Eppendorf tube, and then 137 μl 5 M NaCl and 115 μl CTAB/NaCl solution (100:41 mg/ml) were added and incubated at 65 °C for 30 min with shaking in a Hybaid oven. The supernatant was treated twice with 838 μl of chloroform:isoamyl alcohol (24:1) (Sigma, UK) and DNA was precipitated with 815 μl of isopropanol. The pellet was then washed twice in 1 ml of 70% ethanol, dried for 30 min and re-dissolved in 50 μl DEP-treated sterile water. Quantity and purity of the extracted DNA were assessed using NanoDrop ND-1000 spectrophotometer (Nanodrop, Wilmington, USA).