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INTRODUCTION Isolation of Plant DNA: Plant molecular biology studies often require a simple, rapid, and reproducible method

for preparing DNA from a wide variety of species. A number of factors that can affect the yield and purity of DNA must be considered, including incomplete cell lysis and carryover contamination of carbohydrates, polyphenolics, flavones, and other metabolites. Method described by Doyle and Doyle (1990) DNA isolation method uses the detergent cetyltrimethylammonium bromide (CTAB) to lyse plant cells. After lysis, contaminants are removed by a chloroform extraction step. During extraction, it is essential that the correct salt concentration is used to ensure that contaminants are separated into the organic phase and DNA stays in the aqueous phase. DNA is recovered from the aqueous phase with a subsequent precipitation either by adding alcohol or lowering the salt concentration so that DNA forms insoluble complexes with the

Material And Method:DNA ISOLATION: Soybean seeds were used for the isolation of genomic DNA. The seeds were crushed and seed coats were removed. The remaining powder was homogenized in liquid N2 following the method of Doyle and Doyle (1990) in these steps: 1. 2 g seed were crushed till fine powder, seed coats were removed and the remaining powder was homogenized in liquid N2 with the help of mortar-pestle. 2. The homogenized material was transferred to 20 ml pre-warmed (600C) DNA Isolation Buffer (2X CTAB DNA Extraction Buffer - 100 mM Tris, 20 mM EDTA,

1.4 M NaCl, 2% CTAB and 2 l/ml -mercaptoethanol) in capped polypropylene tubes. 3.Clump was suspended gently and slowly by using spatula. 4.The content was incubated for 1 hr. at 60 0C with occasional mixing by gentle swirling in water bath. 5.After removing from water bath one volume of chloroform: Isoamyl alcohol (24:1) was added and mixed by inversion for 15 minutes to ensure emulsification of the phases. 6.Spin at 15,000 rpm for 15 minutes. 7.The step 5 & 6 was repeated again. 8.After centrifugation aqueous phase was taken and transferred to another tube. 9.Ice cold 0.6 vol. of isopropanol was added to precipitate DNA. 10.DNA-CTAB complex was precipitated as a fibrous network, lifted by Pasteur pipette and was transferred to washing solution. In some cases amorphous precipitation was collected by the centrifugation at 5,000-10,000 rpm for 5-10 minutes at 20 C. 11. 20 ml of 70% alcohol was added to the pellet of DNA and was kept for 20 minutes with gentle agitation. 12.The pellet was collected by centrifugation at 5,000 rpm for 5 minutes at 20C. 13.The step 11 & 12 was repeated again, because palette contained slight impurities. 14.The tubes were inverted and drained on a paper towel. The pellet was dried over-night after covering with parafilm with tiny pores. 15.The pellet was re-dissolved in 2000 l of TE buffer by keeping over night at 4C without agitation.

16.The palette contained the impurities of powder and other debris, as normal problem with the isolation of genomic DNA from seeds. The content was centrifuged at 5000 rpm, for 10 minutes, supernatant was taken and used for further analysis. RESULTS DNA isolation:Total genomic DNA was isolated with the CTAB method described by Doyle and Doyle (1990) and treated with RNase to eliminate RNA. DNA concentration was measured by UV-absorbance method. The integrity of the isolated DNA was verified by visualization of DNA on Agarose gel (0.8%) with DNA standard uncut lambda DNA. A single sharp band was observed for isolated DNA. The quality of DNA was determined as the ratio A260/ A280, which ranged from 1.8 to 1.9, which is indicative of good quality plant DNA. Purification of DNA: The extracted DNA contains the impurities of RNA. RNA was removed by treating the sample with DNase free Rnase procured from fermentas. Protein including RNase was removed by treating with chloroform: Isoamyl alcohol (24:1). The purification was carried out in following steps: 1. 2.5 l of RNase was added to 0.5 ml of crude, DNA preparation (2.5 l of RNase = 25 g of RNase, so treatment was 50 g / ml of DNA preparation). 2. Gently it was mixed thoroughly and was incubated at 37 C for 1 hr. 3. After 1 hr, a mixture of 0.3 - 0.4 ml of chloroform: Isoamyl alcohol (24:1) was added and mixed thoroughly for 15 minutes till an emulsion was formed. 4. The content was centrifuged for 15 minutes at 15,000 rpm.

5. Supernatant was taken avoiding the whitish layer at interface. 6. The DNA was re-precipitated by adding double the quantity of absolute alcohol. 7. To pellet the DNA, the tube was centrifuged for 5 minutes at 5,000 - 10,000 rpm. 1. 2. The pellet was washed with 70% alcohol and dried over night. The DNA was re-dissolved in 500l of TE buffer.

Solution based Genomic DNA Isolation

Random Amplified Polymorphic DNA:


This technique utilizes a single random oligomer primer to amplify genomic DNA taken as template. The amplified fragment profile is generated by agarose gel electrophoresis. The difference in the fragment profile of different genotypes can be used as genetic markers for a variety of genotypic identification, genome mapping and gene tagging. Requirements 1. Taq DNA polymerase (5U/l) 2. Random Decamer primers (15 ng/l) 3. Deoxy nucleotide triphosphates (10 mM) 4. Taq DNA polymerase buffer (10 X). 5. TAE Buffer (50 X)

Result:RAPD analysis was performed with three decamer primers purchased from the University of British Columbia, Vancouver, Canada, USA. The conducted experiment suggests that the

isolated DNA was pure and can be utilized for RAPD analysis. As after primer screening all three primers amplified well and can be used for the analysis of various molecular parameters.

Electrophoresis :Widely used to separate proteins under influence of electric field because they are charged. When an electrical gradient is applied, the molecules migrate towards the electrode with the charge opposite to their own, with the result that the single boundary formed by the mixture is broken into several boundary according to the relative motilities of the mixture. Agarose gel

electrophoresis is a method used in biochemistry and molecular biology to separate DNA, or RNA molecules by size. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis). Shorter molecules move faster and migrate further than longer ones. In Polyacrylamide gel electrophoresis (PAGE) is useful for separating and analyzing complex protein mixture. This.multiphase buffer systems employ two kind of gel in one run: the lower separating and the upper stacking gel. SDS is an anionic detergent, which binds strongly to and denatures proteins. The protein-SDS complex carries net negative charges, hence moves towards the anode and the separation is based only on the size of protein. AGAROSE GEL ANALYSIS The integrity of DNA was judged through gel analysis in following steps: 1. Cast 150 ml agorose gel (0.8%) in 0.5X TBE (Tris Borate EDTA) buffer containing 0.5 g / ml of Ethidium Bromide. 2. 5 l of DNA per sample was loaded in each well. 3. Known amount of uncut Lambda phage DNA was also loaded as control. 4. Electrophoresis was conducted at 50 V for 1 hr. 5. Gel was visualized under UV light using trans illuminator. 6. Presence of single compact band at the corresponding position to phage DNA

Plasmid DNA isolation:The genetic manipulations described above require large quantities of DNA. One of the easiest ways to get large amounts of DNA is to place the desired DNA into bacteria, grow the bacteria, then harvest the bacteria, and isolate the DNA. Bacteria can maintain DNA as plasmids: circles of DNA that usually contain a gene that allows the bacterium to grow in the presence of an antibiotic. There are many different kinds of plasmids commercially available. All of them contain 1) a selectable marker (i.e., a gene that encodes for antibiotic resistance), 2) an origin of replication (which is used by the DNA making machinery in the bacteria as the starting point to make a copy of the plasmid) and 3) a multiple cloning site. The multiple cloning site has many restriction enzyme sites and is used to insert the DNA of interest. The multiple cloning site is usually in the middle of a reporter gene like Lac Z. A commonly used plasmid is pBluescript. The introduction of biologically active recombinant plasmid DNA into bacterial cells holds a key position in all cloning experiments. The transfer of DNA (Plasmid in present case) can be effected by transformation, i.e., by the immediate uptake of naked plasmid DNA by competent cells. Only a fraction of cells are competent to take plasmid DNA at a particular stage of growth. Cultures at late log phage of growth (108 cells /ml) were found to be more efficient for transformation. This stage can be achieved roughly in 2.5 to 3 hour of incubation in a 50 ml culture, inoculated with 0.5 ml of overnight grown culture. A number of transformation techniques have been described which are based on the observations originally made by Mandel and Higa (1970), who noticed that the uptake of lambda DNA by bacteria can be increased considerably in the presence of calcium chloride. Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane. In order to make bacteria take in the plasmid, they must first be made

"competent" to take up in a solution with a high concentration of calcium. DNA can then be forced into the cells by incubating the cells and the DNA together on ice, placing them briefly at 42oC (heat shock), and then putting them back on ice. This causes the bacteria to take in the DNA. The cells are then plated out on antibiotic, IPTG and X-gal containing media.

DIFFERENTIAL LEUCOCYTE COUNT (DLC):-

WBCs are of various kinds - monocytes and lymphocytes among agranulocytes and neutrophils, basophils, eosinophils among granulocytes. For symptoms of certain diseases the physician may recommend a DLC or differential leukocyte count, e.g. increased eosinophil count indicates allergy. Differential leukocyte count means detecting the number of different kinds of leucocytes. For DLC, a blood film is prepared, stained with Giemsa or Leishman stain and the percentage of various kinds of leucocytes calculated.

Material And Method:Procedure of making blood film on the slide: -

(a) A small drop of blood is placed in the central line of a slide about, 1-2 cm from one end. (b) The spreader is placed at an angle of 45o to the slide and then moved back to make contact with the drop. (c) The drop should spread out quickly along the line of contact of the spreader with the slide. (d) Spread by a rapid, smooth, forward movement of the spreader.

(e) The drop should be of such size that the film is 3-4 cm in length.

Qualities of a good blood film: (a) It should not be too thin and the tail of the film should be smooth. (b) There should be some overlap of the red cells diminishing to separate near the tail. (c) The leucocytes in the body of the film should not be badly shrunken.

Fig: - Counting of DLC under the microscope

Staining the Blood Film:- Various dyes (stains) are used to stain the blood film for identifying blood cells. Two of them are Giemsa stain and Leishman stain of which Leishman stain is mentioned below: -

LEISHMAN Procedure

STAIN

[0.15%

in

methylalcohol]

(i) Pour enough stain on the smear to cover it fully. (ii) Allow acting for 2 minutes.

(iii) Add twice the quantity of buffer to the stain. (iv) Avoid overflow and suck the mixture in and out with the pipette to ensure thorough mixing. A scum will form on the smear. (v) Allow the diluted stain to act for 5 minutes. (vi) Wash the smear. (vii) Wipe to clean the back of the smear and allow it to dry. (viii) Observe/examine.

Procedure of Count (a) The cells should be counted using high power or oil immersion lens in a strip running the whole length of the film. (b) The lateral edges of the film are avoided. (c) The film should be inspected from the "head" to the "tail". (d) If less than 100 cells are encountered in a single narrow strip, one or more additional strips should be examined until at least 100 cells have been counted. (e) In patients with very high counts (as in leukemia) the cells should be counted in any well spread area where the cell types are easy to identify.

Result:MICROSCOPIC OBSERVATION:I). GRANULOCYTE (a). Eosinophile

(b).Basophile

(c). Neutrophile

II). AGRANULOCYTES (a).Monocyte

(b) Petit lymphocyte

(c). Grand lymphocyte

Normal Differential Leucocyte Count (Adults) Neutrophils.. 40-75% Lymphocytes 20-45% Monocytes 2-10% Eosinophils... 1-6%

Fig:- Formation of blood cell

Observations :A total of 100 cells were observed out of the various leukocyte cell counts were as follows:Neutrophils Lymphocytes Monocytes Eosinophils Basophils - 56 - 32 -7 -4 -1

Dilution of DNA for PCR:The quantitated DNA was diluted to final concentration of 25 mg / l in TE buffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0).

Protocol:
1. Prepare the reaction mixture by adding the following: Water Buffer dNTPs Primer Enzyme DNA to make total volume to 25 l 2.5 l 0.25 l each 1 l 1U 25-50 ng

1. A master mix is prepared if samples are many where DNA can be added separately. 2. Mix gently and carry out the polymerase chain reaction in the thermal cycler with the following specification:

Cycle

920 C 3.5 min. 370C 1 min. 720 C 2 min.

40 cycles:

920C 1 min. 370C 1 min. 720C 2 min.

Last cycle:

720C 7 min.

4. Prepare 1.5% agarose gel in 1 x TAE buffer. 5. Add 2.5 l loading dye to each tube. 6. Load the samples and carry out electrophoresis at 40 V for four hours. 7. Stain gel with ethidium bromide (@ 0.5 g/ml) 8. View gel under UV light. 9. Score the parents for polymorphism. 10. Observe the segregation of polymorphic bands in the F2 plants.

SDS-PAGE ANALYSIS:
Stock solution 1. Acrylamide stock (30%): Dissolve 29.2 g acrylamide and 0.80 g N'N'-Methylene bisacrylamide in water and make up the volume to 100 ml. sterilize the solution by filtration, check the pH of the solution is 7.0 or less, and store the solution in dark bottles at room temperature. 2. 0.5 M Tris-Cl pH 6.8: Dissolve 60.57 g in 800ml of ddH2O. Adjust the pH to 6.8 with HCl and make up the volume to 1000 ml. 3. 1.5 M Tris-Cl pH 8.8: Dissolve 181.71 g in 800ml of ddH2O. Adjust the pH to 8.8 with HCl and make up the volume to 1000 ml. 4. Tris-glycine (running) buffer, (0.025 M Tris, 0.192 M glycine): Dissolve 3.025 g Tris. 14.400 g glycine and 1.0 g SDS in water and make up final volume to 1000ml. 5. Ammonium persulphate solution (10%): Dissolve 10 mg APS in 100 l ddH2O. Make fresh each time. Working solution 1. Resolving gel solution: (10% - acrylamide) (for to 1.5 mm gels) 30% Acrylamide stock 0.75 M Tris, pH 8.8 DDW TEMED 16.6 ml 12.4 ml 20.9 ml 25 l

10% APS Mix well and pour.

150 l

2. Stacking gel solution (3.2 %-acrylamide) (for two 1.5 mm gel) 30% Acrylamide 0.25 M Tris-SDS stock pH 6.8 DDW TEMED 10% APS Mix well and pour Note: Unpolymerized acrylamide is a potent neurotoxin and is absorbed through skin. Avoid skin contact and inhalation by wearing gloves and using fume cupboard. Although polyacrylamide is considered to be non-toxic, but should be handled carefully because of the possibilities that it might contain small quantities of unpolymerized acrylamide. 3. Staining solutions Commassie Blue G 250 Methanol Acetic acid ddH2O 4. Destaining Solution Methanol Acetic acid ddH2O 50 ml 70 ml 880 ml 0.5 g 250 ml 10 ml 240 ml 1.6 ml 1.9 ml 4.0 ml 6 l 100 l

Procedure :1. Clean and dry the glass plates and spacers, then assemble them properly. 2. Degas the resolving gel solution (without ammonium per sulphate) using a vacuum for 35 min and then add ammonium per sulphate solution. 3. Mix gently and carefully, pour the gel solution in the chamber between the glass plates, leaving the space for stacking gel. Layer distilled water or isobutanol on the top of the gel and leave to set for 60 min. 4. Degas stacking gel solution (without ammonium per-sulphate) and then add ammonium per sulphate solution. 5. Remove the water from the top of the gel. Pour the stacking gel mixture, place the comb in the stacking gel and allow the gel to set. 6. After the stacking gel has polymerized, remove the comb without distorting the shapes of the well. Wash the wells with distilled water using a syringe. 7. Prepare samples to be loaded in electrophoresis. 8. Fill the upper and lower tank with electrode buffer and connect upper through to cathode (-) and lower trough to anode (+). Turn on the current to 36 mA for initial 0.5 hr until the sample travel through the stacking gel. Then continue the run at 56 mA until the bromophenol blue reaches the bottom of the gel (about 3 hrs.) 9. After the electrophoresis carefully remove the gel from the glass plates. 10. Then put it in the staining solution for overnight. 11. Destain the gel with several changes of destaining solution till the background becomes clear.

Result:SDS PAGE analysis: The purity of the protein can be determined by the number of bands obtained in lane. Protein sample 1 is serum and contains several bands as serum contains several proteins. Protein sample 2 is Bovine serum albumin and shows a single band. Protein sample 3 is ovalbumin and shows a single band. Protein standard marker has four bands and the molecular weights are given in the table below:

Fig: Appearance of protein bands after SDS-PAGE