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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.
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 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.
18.104.22.168 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]