The fresh young leaf samples with lesser signs of necrosis and chlorophyll accumulation must be collected. Depending upon the distance from field to Lab, either liquid nitrogen or ice packs (4°C) must be used for shipping. In both the cases, the leaves are chopped into smaller pieces (5mm2) removing the major veins and mid-ribs with a sharp sterilized blade (autoclave 121°C, 20 min) on ice in a glass petri-dish (90x100mm2). Use autoclaved distilled water (ADW; chilled at 2°C) during chopping and keep it at 4°C for 3 hrs. The ADW must be changed after every 1 hrs to remove the resin oozed out from leaf parts. Maintain this process in dark to consume the starch inside leaf as well. This is followed by drying the chopped leaves on sterilized filter paper and immediate grinding in liquid nitrogen to make a fine powder. Notice a change in color from green to off-white during grinding in liquid nitrogen. Take all the powder in 50ml falcon tube and immediately stored at -80°C until further analysis.
Preparation of DNA extraction buffer
The Optimized extraction buffer consisted of 100 mM Tris-HCL (PH 7.5), 25 mM EDTA, 1.5 M NaCl, 2.5% (w/v) CTAB, 1.5% PVP, 0.5% L. ascorbic acid, 0.5% BSA and 0.5% (v/v) β-mercaptoethanol). Firstly, the buffer containing 100 mM Tris-HCL (PH 7.5), 25 mM EDTA and 1.5 M is to be autoclaved and stored at room temperature (24°C). Just before use, 2.5% (w/v) CTAB, 1.5% PVP, 0.5% L. ascorbic acid, 0.5% BSA and 0.5% (v/v) β-mercaptoethanol) is added to the pre-quantified volume of the buffer (for each 10ml Extraction should be prepared) and was warmed to 65°C in autoclaved bottles. CTAB, 2.5% w/v and PVP, 3.0% w/v were added and allowed to dissolve by gentle intermittent swirling.
Steps of DNA extraction process
1. Take one gram of frozen finely powdered leaf tissues in a new 50 mL Falcon tube and mixed with the pre-heated extraction buffer (10 ml) for one sample. Breifly, vertex for 30sec to ensure leaf material is fully mixed with buffer. Fine grind is a key to obtaining high DNA quantity with lesser artifacts of resin.
2. If in step one the sample is not grinded with mortar and pestle then vortex the falcon tube for 5 min otherwise proceed to step 3.
3. The falcon tube was kept into the 65°C incubator or water bath and mix gently by inversion after every 10 min till 45 min. After incubation, place the tube at room temperature for five min to reach to room temperature environment. Centrifuge the 50ml falcon tube for 5 min at 3000×g on room temperature. For next generation sequencing, the greater the genome size, lower is speed of initial centrifugation.
4. Transfer 1ml of supernatant to each 2ml Eppendorf tubes already containing 1 ml of chloroform: isoamyl alcohol (24:1). Mix supernatant and chloroform: isoamyl alcohol by gentle inversions for 10 min and subsequently place the tube on ice for 10 min.
5. Centrifuge the tube for 10 min at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Place the tube for 30 min at 37°C.
6. After RNAse A treatment, add one volume of chloroform: isoamyl alcohol again and mix by inversions for 5 min. The tube is centrifuged for 10 min at 5000 × g at 4°C. Transfer the clear supernatant into new 2ml tube and add half volume of 5 M NaCl to the sample and mix gently by inversions.
7. Add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 min in -20°C freezer. It should not be for more time as some remaining phenolics and resin may also precipitate with DNA.
8. After incubation, the tubes were centrifuged at 5000 × g for 10 min at 4°C and the supernatant was gently removed. The pellet is washed two times with 1ml of 70% ethanol and the DNA is pellet by 5000 × g at 4°C for only 5 min. The supernatant is discarded and the pellet is air-dried (10 min). The pellet are allowed to re-suspend in 50 μL of TE (10 mM Tris. HCl pH 8.0; 1 mM EDTA pH 8.0).
DNA extracted by protocol
The comparison of the extracted genomic DNA by the current protocol with already published protocols as mentioned in abstract section was performed. For analyzing, the quality and quantity agarose gel electrophoresis (0.1% agarose) and Qubit 3 Fluorometer were used, respectively (Table 1 and Fig. 1). Briefly, the concentration of extracted DNA B. sacra and B. elongata was 71.046 μg g-1 leaf and 51.05 μg g-1 leaf samples, respectively. The extracted DNA concentration for B. sacra and B. elongata were 2.51 μg g-1 leaf and 0.62 μg g-1 and 5.09 μg g-1 leaf and 2.4 μg g-1 leaf based on the previously published protocols of Bipin Deochand Lade and I. Haque respectively. As shown in Fig. 1, the analysis of the extracted DNA on 1% agarose gel electrophoresis appeared high quality due to the single and pure band.