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Holy and sweet basil plants were grown at the National Plant Protection Experimental Station Réduit (Ministry of Agro-Industry & Food Security). All the necessary conditions such as nutrients and pesticides were given to the plants for good growth and development of healthy plants.
Young, tender, unbruised and healthy leaves were picked in the morning from the Experimental Station, kept between moist tissue paper in a plastic bag kept away from sunlight and the fresh samples were brought to the laboratory placed on an ice box for DNA extraction and further analysis.
Experimental analyses have been done on three different types of plant samples,
Leaves of White Tulsi (Ocimum tenuiflorum)
Leaves of Purple Tulsi (Ocimum sanctum)
Leaves of Sweet basil (Ocimum basilicum)
Figure 4: (a) white tulsi light green leaves, (b) purple tulsi: purplish leaves, (c) Dark green leaves of sweet basil.
2.1 Morphological data collection and charaterisation
The morphological data collection was done on field and personal observation based on the following several phenotypic traits:
Type of leaf shape and stalk
Colour of stem
Strength of stem
Color and intensity of leaves
Type of venation
Color of midrib
Length and width of leaves
2.2 Materials and Methods for DNA extraction
Several experiments were carried out which are described below however only the optimized protocol which gives the best DNA bands results were subjected to further analysis for PCR reaction.
2.2.1 DNA Extraction and Purification
Genomic DNA was isolated from O.tenuiflorum, o. sanctum and O.basilicum specimems using three protocols: the modified hexadecyltrimethylammonium bromide (CTAB) mini preparation described by Doyle and Doyle 1990, with 1% 2-mercapthoethanol as a reductant, the modified sodium dodecyl (SDS) mini preparation method of Edwards et al. (1991) with 1% 2-mercapthoetnahol as reductant and using the modified Dellaporta and Doyle &Doyle protocol method which are described below.
The modified Dellaporta and Doyle & Doyle protocol was used for DNA extraction.
1 ml of CTAB extraction buffer was placed in a 1.5 ml eppendorf tube and incubated at 60 Â°C water bath for 10 minutes (5 replicates were done for each leaf sample).
0.15 g of fresh young leaf tissue was ground with a pestle and mortar in liquid forming a fine powder.
The grindate was quickly transferred into the eppendorfs containing the pre - heated CTAB buffer.
2 Î¼l of 0.2 %( v/v) b-mercaptoethanol and 2 Î¼l of 0.2 % (v/v) PVP were added to the mixture.
The eppendorf tubes were incubated in 60 Â°C water bath for 25-30 minutes with occasional swirling every 10 minutes.
After incubation, the eppendorf tubes were removed from the water bath.
660 Î¼l of chloroform: isoamyl alcohol (24:1) was added.
The tubes were inverted several times before centrifuged at 10,000 rpm for 10 minutes.
The supernatant was removed with a micropipette and placed into another eppendorf tube.
Steps 7-9 were repeated.
660 Î¼l of ice-cold isopropanol was added and the tubes were placed in -20Â°C freezer for further DNA precipitation (left overnight).
The following day, the tubes were centrifuged for 30 minutes at 12,000 rpm.
The supernatant was poured off, and the DNA pellet was washed with 70% ethanol (tubes spinned at 10,000 rpm for 5 minutes).
The alcohol was poured off and the DNA pellet was allowed to completely dry in the centrifugal evaporator for 20 minutes.
Finally, the DNA pellet was dissolved in 50 Î¼l sterile distilled water.
CTAB mini preparation method with 1% 2-mercaptoethanol
The leaves were weighed to about 500 mg per tube into an eppendorf tube and then dropped in liquid nitrogen for 2 min.
The weighed leaves were ground in 600 ml extraction buffer (100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl, 2% CTAB), and 1% 2-mercaptoethanol) preheated to 65Â°C and incubated for 45 min at 65Â°C and vortexed every 15 min.
The mixture was homogenized with an eppendorf homogeniser and added about 10 - 20 mg of polyvinylpolypyrrolidone (PVPP). (Modification was made here instead of PVPP, PVP was used)
500 ml of chloroform: isoamylalcohol (24:1) was added and mixed by constantly swirling for 10 min and centrifuging for 5 min at 14,000 rpm.
The supernatant was removed to a clean tube.
The previous step was repeated and the supernatant was transferred again to a clean micro tube.
The nucleic acids were precipitated by addition of 600 ml ice cold isopropanol and centrifuging at 14,000 rpm for 5 min. The DNA pellet was washed with 1000 ml cold 70% ethanol. The pellet was then dried and later resuspended in 100 ml sterile distilled water and put in water bath overnight at 55Â°C.
(c) SDS mini preparation method with 1% 2-mercaptoethanol
The leaves were weighed to about 500 mg per tube into an eppendorf tube and then dropped in liquid nitrogen for 2 min.
The weighed leaves were ground in 400 ml extraction buffer (200 mM Tris Hcl pH 7.5, 25 mM EDTA pH 8.0, 250 mM NaCl, 10% SDS (sodium dodecyl sulphate), and 1% 2-mercaptoethanol).
The mixture was homogenised with an eppendorf homogeniser and added about 10-20 mg of polyvinylpolypyrrolidone (PVPP).
Another 400 ml extraction buffer was added and the homogenate vortexed followed by centrifuging for 2 min at 14,000 rpm in order to pellet the plant debris.
The supernatant was removed to a clean tube. An equal volume of chilled phenol: chloroform: isoamylalcohol (25:24:1) was added.
The samples were mixed well to emulsify and then centrifuged at 10 000 rpm for 10 min.
The nucleic acids were precipitated by addition of 600 ml ice cold isopropanol and centrifuging at 14,000 rpm for 5 min.
The DNA pellet was washed with 1000 ml cold 70% ethanol. The pellet was then dried and later resuspended in 100 ml sterile distilled water and put in water bath overnight at 55Â°C.
2.2.2 Purification of DNA for the mini CTAB and SDS preparation
The samples isolated using the above methods were purified as detailed. To each tube, 500 ml chloroform: iso-amylalcohol (CIA 24:1) was added and the contents mixed by shaking for 15 min, followed by centrifugation at 12000 rpm for 15 min. The aqueous phase was transferred to a new tube and then 200 ml 1M NaCl-TE added to the old tube and shaken for 15 min. The old tube was centrifuged for 15 min at 12000 rpm. The aqueous phase was transferred to the new tube and mixed, followed by centrifugation at 12000 rpm for 15 min in order to settle any remaining debris. The supernatant was then transferred to a new tube. Ice cold isopropanol (700 ml) was added to the sample and mixed gently, and centrifuged at 10,000 rpm for 5 min and the supernatant discarded. Cold 75% ethanol (1000 ml) was added to the pellet to wash it thrice, and contents centrifuged at 5000 rpm for 5 min. The ethanol was discarded and the pellet air dried. The pellet was re-suspended in 200 ml sterile distilled water (SDW) and incubated overnight at 55Â°C.
NOTE: Modifications were brought to procedure for the extraction of DNA with the mini CTAB and SDS protocol as well for its purifications; the overnight incubation was not done instead after adding the sterile distilled water the pellet was kept frozen at -20Â°c overnight and was then subjected for further analysis.
220.127.116.11 RNase treatment for the mini CTAB and SDS preparation
The DNA was treated with DNase free Ribonuclease A (10 mg/ml). Large amounts of RNA in the sample can chelate Mg2+ and reduce the yield of the PCR (Padmalatha and Prasad, 2006). This step removes RNA from the isolated genomic DNA. RNase (10 Î¼l of 10 mg/ml; Sambrook et al., 1989) was added to 100 Î¼l of re-suspended DNA pellet and then incubated at 37°C over night. Equal volume of ice-cold absolute ethanol was added to each sample and then centrifuged at 10,000 rpm for 10 min to re-precipitate the DNA. This was done twice. The supernatant was poured off and the DNA pellets air-dried and re-suspended in 100 Î¼l double sterile distilled water (dSDW).
2.3 RNASE TREATMENT OF GENOMIC DNA
100 Î¼l (0.1 ml) of DNA was put in an eppendorf tube (6 eppendorf tubes in total each containing DNA sample of respective basil variety).
1 Î¼l RNAse was added in the eppendorf containing the DNA and the mixture was incubated at 37 Â°C for 1 hour.
10 Î¼l of 3M sodium acetate was then added.
100 Î¼l of phenol: chloroform:isoamyl (25:24:1) was added and mixed well by inverting the eppendorf tube.
The eppendorfs were spinned at 10,000 rpm for 5 minutes.
The supernatant was collected and 100 Î¼l of chloroform:isoamyl(24:1) was added.
The eppendorfs were spinned again at 10,000 rpm for 5 minutes.
The supernatant was collected into another clean eppendorf and 100 Î¼l of cold isopropanol was added (left overnight at -20 Â°C for DNA precipitation)
The eppendorfs were centrifuged at maximum r pm for 30 minutes after which the isopropanol supernatant was discarded.
The DNA pellet was washed with 70 % alcohol and the pellet was dried in the centrifugal evaporator.
The DNA pellet was dissolved in 100 Î¼l sterile distilled water.
Samples were stored in -20Â°c.
2.4 Evaluation of quality and quantity of DNA
Quality Assessment of DNA
In the experiment carried out, 10 Î¼l DNA was mixed with 990 Î¼l sterile distilled H2O in a quartz cuvette for each variety and the absorbance of DNA was read at wavelengths 230, 260 & 280 nm respectively in the spectrophotometer. The absorbance reading of all the 3 species were taken and the purity and quantity of isolated DNA were determined spectrophotometrically.
Wavelength ratios showing quality of DNA
OD260/OD280 =1.8 or 1.9 Pure DNA
OD260/OD280 >1.8 DNA contaminated with RNA
OD260/OD280 <1.8 Phenol or protein contamination
OD260/OD230 <1.8 Polysaccharides or starch contamination
Quantity determination of DNA
The formula used to calculate the DNA concentration:
DNA concentration (Î¼g/Î¼l) = Optical density value at 260 nm x 0.05 x dilution factor.
The dilution factor is 1000 divided by 10, since 10 Î¼l DNA was diluted with 990 Î¼l Sterile distilled water making a total volume of 1000 Î¼l.
2.5 Electrophoresis Analyses
The DNA samples were mixed with the gel loading buffer and loaded onto a 1.5% agarose gel and left to migrate for about one hour at 90 volts. The volumes used were 7Î¼L of DNA and 3Î¼L of Dye. The DNA was then stained with Ethidium Bromide and viewed under UV light.
2.6 Preparation of an Agarose gel medium of 1.5% concentration (See Appendix).
2.7 RAPD Marker Analysis
A set of 12 primers ( OPK-05; OPL-05; OPO-03; OPC-08; OPW-04; OPC-03; OPC-16; OPP-20; OPA-18; OPA-10; OPB-11 and OPD-13) were used.
The DNA sample was diluted from the stock with nanopure water making up 50 Î¼l and placed on ice.
Dilution of DNA sample = 20 ng/ Î¼l x 50 Î¼L + dilution with nanopure water
[DNA] 260 nm
Table 5. Optimisation Protocol for RAPD Reaction mixture
Volume per reaction tube (Î¼l)
Volume for 3 tubes (Î¼l)
The master mix was prepared on ice for a total of 3 PCR tubes as follows:
Two PCR tubes were used for each primer:
1st PCR tube: Positive control which contained 2 Î¼l of the diluted DNA.
2nd PCR tube: Negative control which contained no DNA.
2.8 Detailed Steps of PCR (See Annex)
DNA amplification was carried programmed with 3 min at 94Â°C for initial denaturation, followed by 35 cycles of 54sec at 94Â°C, 45 sec at 43Â°C, 2 min at 72Â°C, and a final 5 min extension at 72Â°C. After amplification, the DNA fragments were separated by electrophoresis for about 3hours under constant voltage (90 V) in 1.5% agarose gel submersed in 1X TBE buffer. The gels were stained with ethidium bromide solution and observed under ultraviolet light. A 1 kb fragment size marker was used as a reference to allow comparison among the different gels (1kb ladder).
2.9 Different molarities of Template DNA were used for screening of primers
Volume of diluted DNA per
reaction tube [Î¼l]