GeNei TM Genomic DNA Extraction. Teaching Kit Manual. GeNei TM. Cat No. New Cat No. KT KT67A Genomic DNA Extraction (From Leaves)

Transcription

1 Genomic DNA Extraction (From Leaves) Teaching Kit Manual Cat No. New Cat No. KT KT67A Revision No.:

2 CONTENTS Page No. Objective 3 Principle 3 Kit Description 6 Materials Provided 7 Procedure 8 Observation & Interpretation 10 AGAROSE GEL ELECTROPHORESIS Introduction 13 Principle 13 Procedure 16 ORDERING INFORMATION 18 1

3 Objectives: To extract genomic DNA from leaves. To analyze the extracted DNA by agarose gel electrophoresis. Principle: Plants contain three types of DNA: nuclear, mitochondrial and chloroplast. Although quite elaborate methods exist for the isolation of each type of DNA, most experiments require only a simple preparation of total DNA. The two main problems in isolating DNA from plants are the presence of DNases that degrade the DNA and the presence of other macromolecules (polysaccharides, polyphenols) that co-purify with or polymerize to the DNA during isolation procedure. Three major types of techniques or combination of them are employed in isolation of nucleic acids: differential solubility, adsorption methods or density gradient centrifugation. Choice of method depends on type of DNA being isolated and the application. Major goal of nucleic acid isolation is removal of proteins, which is accomplished due to their different chemical properties. Most nucleic acid isolation protocols involve: Cell lysis step Enzymatic treatments Differential solubility (phenol extraction or adsorption to solid support) Precipitation. 2 3

4 Cell lysis: Nucleic acids (DNA / RNA) must be solubilized from cells or other biological material. This solubilization is usually carried out under denaturing conditions such as SDS, alkali, boiling or chaotropic agents. These denaturing conditions efficiently solubilize the nucleic acids and generally do not adversely affect them. In addition, the denaturing conditions promote the removal of proteins during subsequent steps and inhibit the activity of nucleases that degrade the nucleic acids. Precipitation: DNA is precipitated from dilute solutions with ethanol or isopropanol, in presence of sodium or potassium acetate, ph , added to a final concentration of 0.3 M. Sodium and acidic ph will neutralize the highly charged phosphate backbone and promote hydrophobic interactions. The precipitated DNA is collected by centrifugation or is spooled out with a pasteur pipette. The pellet is rinsed with 70% ethanol to remove any excess salt, dried and dissolved in an appropriate buffer. Enzymatic treatment: Unwanted components (like proteins and RNA) are degraded by enzymes, generally included in lysis buffer i.e., Proteases (Proteinase K): To remove proteins. RNases (RNase A): To get rid of RNA. Differential solubility: Phenol Extraction: Phenol is an organic solvent used to separate proteins from DNA. It is mixed in equal volumes with the DNA. The two phases are then separated by centrifugation and the upper aqueous phase that contains the nucleic acid is retained. Proteins are seen as flocculent material at the interface. Adsorption to solid support: DNA is known to adsorb to silica gel surfaces in presence of high concentration of chaotropic salts that remove water from hydrated molecules in solution. Polysaccharides and proteins do not adsorb to silica gel and are thus removed. DNA is then eluted out under low-salt conditions, free of RNA. 4 5

5 Kit Description: Students will use the lysis buffer supplied to extract genomic DNA from leaves. Lysis buffer when incubated with the plant cells at higher temperature dissolves the cell wall, degrades proteins and inhibits DNase activity. Upon centrifugation, cell debris and other proteins will be separated. The resulting supernatant which consists mainly of genomic DNA and RNA, will be precipitated using alcohol, dried and rehydrated in an appropriate buffer. Quality of isolated DNA will then be analysed by electrophoresing along with control DNA supplied, on a 1% agarose gel. KT67 : The kit is designed to carry out 5 sets of experiments. Each set of experiment consists of 5 DNA extractions from leaves. The kit also includes electrophoresis equipment (ETS-1) required for agarose gel electrophoresis. KT67A : The kit is designed to carry out 5 sets of experiments. Each set of experiment consists of 5 DNA extractions from leaves. Note : Electrophoresis equipment is required for KT67A. Duration of experiment: Approximately 5-6 hours. Materials Provided: The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 6 months of arrival. Quantity Materials KT67/67A Store (5 sets of expts.) Solution A 15 ml 4 C Solution B 1.25 ml 4 C Sodium acetate 1.25 ml 4 C Alcohol 30 ml 4 C Control DNA (ready to use) 50 µl 4 C 2.5X Gel loading buffer 0.25 ml 4 C 6X Staining dye 40 ml RT Agarose 2.5 g RT 50X TAE 20 ml RT 1.5 ml vials 50 Nos. RT Materials Required: Equipment : Freezer, Microcentrifuge, Rocker (optional), Vortex mixer. Glassware : Beaker, Conical flask, Measuring cylinder, Staining tray. Reagent : Distilled water (Sterile). Other Requirements : Leaves (young and tender), Tips, Micropipette, Thermometer, Water bath. 6 7

6 Note: Read the entire procedure before starting the experiment. Collect young tender leaves from any plant source. eg. Papaya, Mango, Rice, Cotton, Hibiscus, Mulberry, Chilli etc. DNA yield will vary depending on the source. Carry out 5 extractions at a time. Prepare required amount of 70% alcohol using distilled water, just before use. Control DNA supplied is ready to use and can be loaded directly onto agarose gel. For preparation of agarose gel, staining and destaining, refer Agarose gel electrophoresis. Procedure: 1. Wash the leaf with sterile water and rinse it with alcohol. Take around 10 to 15 mm leaf disc and place it in a 1.5 ml sterile vial. 2. Crush the leaf into fine pieces with a sterile tip. 3. Add 500 µl of pre-cooled solution A and crush again till the soup is green, this indicates cell lysis. 4. Transfer the vials to a water bath maintained at C and incubate for 5 minutes. Crush the leaves to release more green pigment and further incubate at 70 C in the water bath for minutes. 5. Vortex for 5 seconds and spin at 10,000 rpm for 20 minutes at room temperature. 6. Take the supernatant and add 1 ml of cold alcohol and 50 µl of sodium acetate. Mix gently by inverting the vial and incubate at 20 C for 60 minutes. 7. Centrifuge the vials at 12,000 rpm for 30 minutes at room temperature, drain out the supernatant completely and blot dry. Care should be taken not to dislodge the pellet. A white/pigmented particulate matter sticking to the side of the vial indicates precipitated DNA. 8. Add 100 µl of 70% alcohol and spin at 12,000 rpm for 5 minutes. drain out the supernatant completely and blot dry. 9. Suspend the DNA pellet in minimum amount of solution B (20 to 50 µl). Note: Add the solution along the sides of the vial. 10. Incubate the vial at 55ºC for 3-5 minutes for complete solubilization. 11. Spin at 12,000 rpm for 10 minutes to remove any insoluble material and collect the supernatant in a sterile 1.5 ml vial. 12. Add µl of gel loading buffer to each of the freshly extracted DNA samples (5 numbers). 13. Load the samples along with 10 µl of ready to use control DNA on a 1% agarose gel. (note down the order in which the samples have been loaded). 14. Electrophorese the samples at volts for 1-2 hours. 15. Stain with 1X staining dye. 16. Destain to visualize the band(s). 8 9

7 Observation: Examine the band(s) of isolated DNA samples and compare it with control sample. Interpretation: From the gel, one can observe that genomic DNA being high molecular weight runs above the control DNA band, which is approximately 50 kb in size. If shearing has occurred during extraction, one observes DNA band(s) below the control DNA. If RNA is present along with the extracted DNA, it will be seen migrating faster as it is a smaller molecule compared to DNA. (Refer figure 1). 1 2 Agarose Gel Electrophoresis DNA Lane 1 : Leaf genomic DNA Lane 2 : Control DNA ~ 50 Kb RNA Fig 1 : Genomic DNA from leaf run on 1 % agarose gel (stained with EtBr) 10 11

8 Introduction: Agarose gel electrophoresis is a procedure used to separate DNA fragments based on their molecular weight and is an intrinsic part of almost all routine experiments carried out in molecular biology. The technique consists of 3 basic steps: Preparation of agarose gel Electrophoresis of the DNA fragments Visualization of DNA fragments Principle: Preparation of Agarose Gel: Agarose is a linear polymer extracted from seaweeds. Its basic structure is shown in the figure. HO CH2 O HO OH Fig: Basic unit structure of agarose. Purified agarose is a powder insoluble in water or buffer at room temperature but dissolves on boiling. Molten solution is then poured into a mould and allowed to solidify. As it cools, agarose undergoes polymerization i.e., sugar polymers cross-link with each other and cause the solution to gel, the density or pore size of which is determined by concentration of agarose

9 Electrophoresis of DNA fragments: Electrophoresis is a technique used to separate charged molecules. DNA is negatively charged at neutral ph and when electric field is applied across the gel, DNA migrates towards the anode. Migration of DNA through the gel is dependent upon: 1. Molecular size of DNA 2. Agarose concentration 3. Conformation of DNA 4. Applied current Matrix of agarose gel acts as a molecular sieve through which DNA fragments move on application of electric current. Higher concentration of agarose gives firmer gels, i.e., spaces between cross-linked molecules is less and hence smaller DNA fragments easily crawl through these spaces. As the length of the DNA increases, it becomes harder for the DNA to pass through the spaces, while lower concentration of agarose helps in movements of larger DNA fragments as the spaces between the cross-linked molecules is more. The progress of gel electrophoresis is monitored by observing the migration of a visible dye (tracking dye) through the gel. Two commonly used dyes are Xylene cyanol and Bromophenol blue that migrate at the same speed as double stranded DNA of size 5000 bp and 300 bp respectively. These tracking dyes are negatively charged, low molecular weight compounds that are loaded along with each sample at the start of run, when the tracking dye reaches towards the anode, run is terminated. Visualization of DNA fragments: Since DNA is not naturally coloured, it will not be visible on the gel. Hence the gel, after electrophoresis, is stained with a dye specific to the DNA. Discrete bands are observed when there is enough DNA material present to bind the dye to make it visible, otherwise the band is not detected. The gel is observed against a light background wherein DNA appears as dark coloured bands. Alternatively, an intercalating dye like Ethidium bromide is added to agarose gel and location of bands determined by examining the gel under UV light, wherein DNA fluoresces. Note: Ethidium bromide must be handled carefully as it is a mutagen and a carcinogen. Wear gloves while handling EtBr solution & gels stained with EtBr

10 Procedure: Preparation of 1% Agarose Gel 1. Prepare 1X TAE by diluting appropriate amount of 50X TAE buffer. (For one experiment, approximately 200 ml of 1X TAE is required. Make up 4 ml of 50X TAE to 200 ml with distilled water). 2. Weigh 0.5 g of agarose and add to 50 ml of 1X TAE. This gives 1% agarose gel. 3. Boil till agarose dissolves completely and a clear solution results. 4. Meanwhile place the combs of electrophoresis set such that it is approximately 2 cm away from the cathode. 5. Pour the agarose solution in the central part of tank when the temperature reaches approximately 60 C. Do not generate air bubbles. The thickness of the gel should be around 0.5 to 0.9 cm. Keep the gel undisturbed at room temperature for the agarose to solidify. 6. Pour 1X TAE buffer into the gel tank till the buffer level stands at 0.5 to 0.8 cm above the gel surface. 7. Gently lift the combs, ensuring that wells remain intact. Electrophoresis 8. Connect the power cord to the electrophoretic power supply according to the convention red: anode, black: cathode. 9. Load the samples in the wells in the desired order. 10. Set the voltage to 50 V and switch on the power supply. 11. Switch off the power when the tracking dye (bromophenol blue) from the well reaches ¾ th of the gel. This takes approximately one hour. Staining Procedure to Visualize DNA 12. Prepare 1X staining dye by diluting 6X dye (1:6) with distilled water. (Approximately 50 ml of 1X staining dye is required for one experiment. Therefore, make up 8 ml of 6X dye to 48 ml with distilled water). 13. Carefully transfer the gel (from gel tank) into a tray containing 1X staining solution. Make sure that the gel is completely immersed. 14. For uniform staining, place the tray on a rocker for approximately one hour or shake intermittently every 10 to 15 minutes. 15. Pour out the staining dye into a container. (The dye can be reused twice). Destain the gel by washing with tap water several times till the DNA is visible as a dark band against a light blue background. Note: Alternatively, Ethidium bromide can be used for visualizing DNA fragments. Add Ethidium bromide to molten agarose to a final concentration of 0.5 µg/ml (from a stock of 10 mg/ml in water), when temperature is around 50 C. Mix and cast the gel. After electrophoresis, DNA samples can be visualized under UV light, they appear fluorescent. No destaining is required in this case

11 Ordering Information Product Size Cat # Genomic DNA Extraction 1 Pack KT67 Teaching Kit (Consumables for 5 experiments & Elpho Kit (ETS 1)) Genomic DNA Extraction 1 Pack KT67A Teaching Kit (Consumables for 5 experiments) Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com 18 19