Plant material and reagents preparation
To ensure availability of fresh leaf samples, mature abaca variety Abuab (NSIC 2017 Mt 001) was obtained from the PhilFIDA Albay Tissue Culture Laboratory. Ihalas, a wild abaca variety, was collected in the Leyte Province. The collected abaca varieties were housed at the National Institute of Molecular Biology and Biotechnology and Bureau of Plant Industry until sample processing. Leaf samples were collected from the second to the youngest leaf of the abaca plant.
To ensure a nuclease-free environment, all reagents were prepared in nuclease-free water (Ambion™, Invitrogen). Mortar and pestles and consumables were sterilized at 121oC at 15 psi for 15 minutes and dried thoroughly at a drying oven (65oC) prior to use. Stock buffers such as 1.0 M Tris-HCl (pH 8.0 and pH 7.5) (Scharlau), 0.5 M EDTA (pH 8.0) (Scharlau), 5M NaCl (Scharlau), 10% (w/v) CTAB (Sigma), 10% (w/v) (sodium dodecyl sulfate) SDS (Merck), 4M guanidine thiocyanate (Sigma), 5 M sodium acetate (pH 5.2) (Scharlau), 5 M potassium acetate (pH 4.8) (Scharlau) and 10% (w/v) PVP (30K) (Sigma) were prepared with nuclease-free water and sterilized at 121oC at 15 psi for 15 min. Ethanol solutions, isopropanol solutions, 10 mg/mL RNase A (Roche) and 1 mg/mL proteinase K (Roche) were diluted with nuclease-free water and filter-sterilized through 0.22 µm filter. All leaf samples were obtained fresh and were ground into a fine powder using liquid nitrogen in the sterile mortar and pestle prior to homogenization with respective protocols’ extraction buffers. All centrifugation steps were conducted in a refrigerated microcentrifuge (Hermle Z 32 HK) at 4oC unless temperature was otherwise stated. All extraction buffers were preheated to 65oC in a water bath prior to use to prevent precipitation of contents.
DNA Extraction Protocols
Protocol1: CTAB-method
The protocol was based on the CTAB method of Gawel and Jarret [16] with modifications. Briefly, 100 mg of tissue sample was ground into a fine powder using liquid nitrogen and homogenized in 700 µL of CTAB buffer (0.1 M CTAB, 1.4 M NaCl, 0.02 M EDTA, 2% CTAB and 0.2% β-mercaptoethanol added before use). Six hundred microliters of the homogenate was transferred to a 1.5 mL tube and incubated at 65 °C for 45 min with inversion every 10 min. Equal volume (600 µL) of 24:1 chloroform:isoamyl alcohol was added and mixed thoroughly. The mixture was centrifuged at 15,000 × g for 2 min at room temperature (RT), and the resulting supernatant (500 µL) was treated with 0.5 µL of 10 mg/mL RNaseA for 15 min at 37oC in a dry bath (AccuBlock™, Labnet) with constant inversion every 5 min. After RNAse A treatment, chloroform:isoamyl alcohol extraction was repeated. Two volumes (800 µL) of 95% ethanol was added to the recovered supernatant (400 µL) and incubated at − 20 °C for 1 h. The mixture was centrifuged at 15,000 × g for 2 min at 4oC, and the resulting pellet was washed twice with 75% ethanol. The total recovered DNA pellet was resuspended in 30 µL of nuclease-free water.
Protocol 2: CTAB-method With PVP/
The protocol was based on Hossain et al. [17] with modifications. One hundred milligrams of tissue sample was ground into a fine powder using liquid nitrogen, and 800 µL of CTAB buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM EDTA pH 8.0, 2% w/v CTAB, 1% w/v PVP, 0.2% v/v β-mercaptoethanol added before use) was added to the ground tissue and homogenized. The homogenate (700 µL) was transferred to a 1.5 mL tube and was incubated at 65oC for 15 min with constant inversion every 5 min. Tubes were centrifuged at 15000 × g for 5 min at RT, and the resulting supernatant (600 µL) was transferred into a 1.5 mL tube. Equal volume (600 µL) of 25:24:1 phenol:chloroform:isoamyl alcohol was added to the supernatant and mixed by inversion and vortexing. The mixture was centrifuged at 15000 × g for 5 min at 4oC and the supernatant was recovered (500 µL) and treated with 0.5 µL of 10 mg/mL RNaseA for 15 min at 37oC with constant inversion every 5 min. Chloroform:isoamyl alcohol extraction was repeated and 50 µL of 5 M sodium acetate (pH 5.2) was added to the supernatant, followed by subsequent addition of ice cold absolute ethanol (900 µL). Tubes were then incubated at -20oC for 20 min, and DNA was pelleted through centrifugation at 15000 × g at 4oC for 5 min. The DNA pellet was recovered through decantation of the absolute ethanol and was further purified through washing twice with 600 µL of 70% ethanol. Finally, the pellet was resuspended in 30 µL of nuclease-free water.
Protocol 3: CTAB-method With 0.3% V/v β-mercaptoethanol
The protocol was based on Healey et al. [14] with minor modifications. One hundred milligram of tissue sample was ground into a fine powder using liquid nitrogen, and 800 µL of CTAB DNA extraction buffer (100 mM Tris-HCl pH 7.5, 25 mM EDTA pH8.0, 1.5 M NaCl, 2% w/v CTAB) supplemented with 0.3% (v/v) β-mercaptoethanol was added. The mixture was further homogenized and was transferred to a 1.5 mL tube. Tubes were incubated at 65oC for 30 min with constant inversion every 10 min. Solid debris were separated from the liquid portion through centrifugation (15000 × g for 5 min at RT). The resulting supernatant (600 µL) was transferred to a new 1.5 mL tube and an equal volume (600 µL) of 24:1 chloroform:isoamyl alcohol was added. The mixture was constantly inverted for 5 min to fully mix the solution and the aqueous phase was separated by centrifugation (15000 × g for 5 min at 4oC). Five hundred microliters of the recovered aqueous phase was transferred to a new 1.5 mL tube and RNA was digested with the addition of 0.5 µL of 10 mg/mL RNAse A. The mixture was incubated at 37oC in a dry bath for 15 min with inversion every 5 min. Chloroform:isoamyl alcohol extraction was repeated, and 400 µL of the resulting supernatant was divided among two 1.5 mL tubes. DNA was precipitated by addition of ½ volume (100 µL) of 5 M NaCl and 3 volumes (600 µL) of ice cold 95% ethanol and incubation at -20oC for 30 min. Tubes were centrifuged at 15000 × g for 5 min at 4oC to pellet the DNA precipitates. Pellets were washed twice with 600 µL of 70% ethanol (centrifuged at 15000 × g for 5 min at 4oC) and were resuspended in 15 µL of nuclease-free water.
Protocol 4: SDS-method
The protocol was based on Ihase et al. [18] with modifications. One hundred milligram of leaf sample was powderized using liquid nitrogen and homogenized in 800 µL of extraction buffer (100 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 5% w/v SDS). The homogenate was transferred into a 1.5 mL tube and was centrifuged at 15000 × g for 5 min at at 4oC. The supernatant (700 µL) was transferred into a 2.0 mL tube and was mixed with 200 µL of 5 M potassium acetate through vortexing. An equal volume (900 µL) of 25:24:1 of phenol:chloroform:isomayl alcohol was added to the mixture, and were altogether mixed thoroughly through vortexing. Mixture was centrifuged at 15000 × g for 5 min at at 4oC. The supernatant (1 mL) was transferred and equally divided between two 1.5 mL tubes and treated with 0.5 µL of 10 mg/mL RNase A for 15 min at 37oC with constant inversion every 5 min. An equal volume (500 µL) of 24:1 chloroform:isoamyl alcohol was added and mixed well by inversion, followed by centrifugation (15000 × g for 5 min at 4oC). The supernatant (400 µL) was transferred to a 1.5 mL tube and was added with 800 µL of absolute ethanol. Tubes were inverted gently and centrifuged at 15000 × g for 5 min at 4oC. The resulting pellet was washed twice with 800 µL of 70% ethanol, air dried and resuspended in 15 µL of nuclease-free water.
Protocol 5: CTAB-method With Triton X-100 And PVP
The protocol was based on Rezadoost et al. [19] with minor modifications. One hundred milligram of leaf sample was powderized using liquid nitrogen and was homogenized with 800 µL of Buffer 1 (200 mM Tris–HCl, 1.4 M NaCl, 0.5% v/v Triton X-100, 3% w/v CTAB). To the homogenate, 0.1% w/v PVP was added and further homogenized. The mixture was transferred to 1.5 mL tube, was vortexed for 20 s and was incubated at 65oC for 30 min. Four hundred microliters of 24:1 chloroform:isoamyl alcohol was added to the mixture, followed by a 2-min inversion of the tubes to mix them thoroughly. Tubes were centrifuged at 15000 × g for 5 min at 4oC. The resulting supernatant (300 µL) was transferred to a 2.0 mL tube, where a half volume (150 µL) of Buffer 2 (50 mM Tris–HCl, 2 M guanidine thiocyanate) with freshly added 0.2% v/v β-mercaptoethanol, 0.2 mg/ml proteinase K and 0.5 µL of 10 mg/mL RNaseA was added and mixed thoroughly by inversion. Mixture was incubated at 40oC for 15 min. After incubation, 2 volumes (600 µL) of 4M NaCl was added and the mixture was placed on ice for 5 min. Two volumes (600 µL) of ice cold isopropanol was added to the mixture and placed on room temperature for 2 min. A pellet was collected by centrifugation at 15000 × g for 5 min at 4oC. The pellet was washed twice with 75% ethanol, air dried and dissolved in 30 µL of nuclease-free water.
Sample Homogenization Through TissueLyserII (Qiagen)
One hundred milligram of cut leaf sample was placed in a 2.0 mL tube together with 2 sterile 7 mm stainless steel beads. Liquid nitrogen was poured and allowed to evaporate until leaf sample was crisp. Frozen samples were ground into fine powder by setting the TissueLyserII (Qiagen) speed at 30 Hz for 1 min. Appropriate amounts of extraction buffer (depending on protocol chosen) were added and samples were further homogenized at 30 Hz for 1 min. Subsequent extraction steps were then followed based on the chosen protocol.
Analysis Of Extracted DNA
DNA (2 µL) was analyzed by agarose gel electrophoresis using 1% (w/v) agarose in 0.5X Tris-Acetate EDTA (TAE) buffer (20 mM Tris base, 10 mM acetic acid, 0.5 mM EDTA). Electrophoresis was performed with 0.5X TAE buffer at a constant voltage of 100 V for 30 min. Gels were stained in a solution of 600X of GelRed™ (Biotium) after the run. The DNA profiles were visualized under ultraviolet (UV) light, and images were acquired with AlphaImager® gel documentation system (ProteinSimple).
The concentration, A260/A280 and A260/A230 ratio were measured with NanoDrop™ 2000c Spectrophotometer (Thermo Scientific) using 2 µL of each sample and with nuclease-free water (Ambion™, Invitrogen) as blank.