Murraya Koenigii Essay

MATERIALS AND METHODS Leaves of Meetha Neem (Murraya koenigii (L.)) were collected from diverse places of Rajasthan. Soft/tender leaves of individual plants were plucked for extraction of DNA for diversity study.

Plant of study:

Kingdom : Plantae

Division : Magnoliophyta

Class : Magnoliopsida

Order : Sapindales

Family : Rutaceae

Genus : Murraya

Species : koenigii Places from where experimental plant material was collected (Semi Arid Eastern Region):

District Place Code 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

EXTRACION OF DNA:

DNA was extracted from tender leaves by CTAB method as given by Doyle and Doyle (1990). The method

Centrifuged for 10 min at 10000 rpm and aqueous phase was transferred to fresh tube.

5. This step was repeated twice.

6. Two third volume of isopropanol was added, DNA allowed to settle.

7. DNA was spooled out in the eppendorf tube and then settle down DNA and add 200 µl of 70% ethanol was added for washing.

8. Centrifuged at 10000 rpm for 10 min, alcohol wash was repeated again.

9. Pelleted DNA was kept for drying for 24 hrs.

10. Dried DNA was dissolved in 300µl of TE buffer.

PURIFICATION OF DNA:

Purification of DNA was carried out by phenol extraction and ethanol precipitation method. Various techniques in molecular studies need DNA of very high purity. After DNA isolation, DNA often contains compounds like polysaccharides, proteins and other cell derbies in presence of these compound it is difficult for the DNA to get amplified thus purification of DNA is needed to get a suitable PCR product.

DURING DNA PURIFICATION 3 PHASES WERE FORMED:

1. The organic phase which consists of RNA and lipid impurity.

2. Interphase consists of protein impurity.

3. The aqueous phase consists of pure DNA.

MATERIALS REQUIRED:

1. RNAse.

2. Phenol: Chloroform: isoamylalcohol (25:24:1) .

3. Sodium acetate

Ensured the complete dissolution of Agarose by checking against the light.

3. Cooled the solution to 50°C and added 4.5 l of ethidium bromide dye to a final concentration of 0.5g/ml and mixed thoroughly.

4. Slowly poured the warm Agarose solution into the casting tray and inserted the required combs.

5. After the gel was completely set the combs were removed carefully and mounted the gel in electrophoresis tank.

LOADING, RUNNING AND VISUALIZATION OF SAMPLES:

1. Electrophoresis tank was filled with the buffer (1X TBE) just enough to cover the gel to a depth of about 1mm.

2. Samples of DNA were mixed with loading dye and loaded the mixture into the wells of submerged gel.

3. Switched on the power pack so that the DNA migrates towards the anode.

4. Applied the appropriate voltage (1-5volt/cm). Run the gel until the Bromophenol blue and Xylene cyanol dye has been migrated to the appropriate distance through gel.

5. Electric current turned off and the gel was visualized and photographed using Kodak gel documentation system. Then photographs were retained for further analysis.

MICROSATELLITE GENOTYPING:

Microsatellite data analysis and Interpretation of data was performed by software package