After collection the fresh leaves sample should be immediately frozen in liquid nitrogen and until further analysis. Before starting DNA extraction take the sample out from liquid nitrogen or -80 and by pressing the frozen leaves it well chopped into small pieces. Put all the leaves material in the beaker containing autoclave distal water around 200 ml. Shake the beaker for 1 minute and the then keep it for 2 to 3 minutes so the midribs of the leaves will set down. Transfer the leaves parts into another beaker which containing same amount of autoclave water and keep it for four hours at 4°C and change the water after each hour. After this, take the leaves out from water and make it air dry for minutes. Grind the leaves into a fine powder in cool mortar and pestle with the help of 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
- Take one 1 gram of frozen leaf tissue (powder already prepared) in a new 50 mL Falcon tube directly and mixed in the pre-heated extraction buffer 10 ml for one sample. At this stage, if the sample is not grinded well previously in the liquid nitrogen can be grind again in mortar and pestle and recover everything again in 50 ml falcon tube.
- If in step one the sample is not grinded with mortar and pestle then vortex the falcon tube for 5 minutes otherwise proceed to step 3.
- The sample was put into the 65°C incubator or water bath and mix by inversion after every 10 min for 45 min. After incubation placed the tube at room temperature for five minutes. Centrifuge the 50ml falcon tube for 5 minutes at 5000 × g on room temperature. Transfer 1ml of supernatant to each 2ml tube already containing 1 ml of chloroform: isoamyl alcohol. Mix supernatant and chloroform: isoamyl alcohol by inversion for 10 minutes and subsequently placed the tube on ice for 10 minute.
- Centrifuge the tube for 10 minute at 5000 × g at 4°C. Transfer the upper aqueous phase into new 2ml tubes and add 5μl RNAse A (10 mg/mL). Placed the tube for 30 minutes at 37°C. After RNAse A treatment add one volume of chloroform: isoamyl alcohol again and mix by inversion for 5 minutes. The tubes is centrifuged for 10 minute 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 inversion. Then, add 2 volumes of cold 95% ethanol and mix gently by inversion. The tubes are incubated at for 45 minutes (should not be more than 1 hour because after long incubation the resin is precipitated with DNA) in -20°C freezer.
- After incubation, the tubes were centrifuged at 5000 × g for 10 minute at 4°C and the supernatant was gently poured off. The pellets were washed two times with 1ml of 70% ethanol and pellet the DNA by 5000 × g at 4°C but for only 5 min. The supernatant was discarded and the pellets were air-dried (10 minute). The pellets were 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 the current protocol
The comparison of the extracted genomic DNA by the current protocol with already published protocols as mentioned in abstract section. For analyzing, the quality and quantity was measured using agarose gel electrophoresis (0.1% agarose) and Qubit 3 Fluorometer, respectively in 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 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 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.