Teaching Kit Manual Cat No. New Cat No. KT43 106279 KT43A 106300 KT43B 106301 Revision No.: 00280507
CONTENTS Page No. Objective 3 Principle 3 Kit Description 5 Materials Provided 7 Procedure 8 Observation & Interpretation 11 AGAROSE GEL ELECTROPHORESIS Introduction 15 Principle 15 Procedure 18 ORDERING INFORMATION 20 1
Objective: To learn the technique of DNA purification from agarose gel using silica. Principle: Electrophoresis through polyacrylamide or agarose gels is one of the core techniques used in molecular biology. These gels are used to separate, identify and purify DNA fragments. Agarose gels can be used to effectively separate fragments from 50 bp to several thousand bases in length by varying the porosity of the gel and application of current. Migration of DNA through the pores of gel depends upon the size and conformation of DNA. DNA can be purified from gels by a number of methods such as: Electroelution, electrophoresis onto DEAE Cellulose/Nitrocellulose (NA45) paper, using β-agarase (from low melting agarose) or using glass beads/ silica etc. In this kit, DNA is purified from agarose gel using silica/ glass powder of a specified size. It is based on the fact that DNA binds to silica under specific conditions of salt and ph. This method works best for purification of fragments between 500 bp to 5000 bp as DNA of smaller sizes (< 500 bp) bind strongly to silica thereby resulting in lower yields, while larger DNA fragments (> 5 kb) tend to get sheared by glass beads. DNA binds to silica in presence of a chaotropic salt, Sodium iodide (NaI) independent of its base composition and topology. Chaotropic salts are known to disrupt the hydrogen bonds of water, thus increasing the solubility of non-polar substances in water. Hence, due to dehydration of phosphodiester backbone by chaotropic salts, the exposed phosphate residues of DNA adsorb to silica. Once adsorbed, double stranded DNA remains in either native/partially 2 3
denatured state and cannot be eluted from the matrix by solvents that displace other biopolymers such as RNA/ carbohydrates/proteins. When rehydrated with aqueous buffer, DNA is eluted out. The purified DNA can be used for any of the following manipulations/operations: Restriction digestion. Ligation Transformation Sequencing Cloning PCR Kit Description: In this kit, students are provided with puc/taq I digest, which has 3 fragments of sizes 1444 bp, 736 bp and 476 bp. These fragments will be separated by electrophoresis, excised out of the gel and purified individually using silica. Each purified fragment will then be analyzed on agarose gel, along with puc/taq I digest. KT43 : Kit is designed to carry out 5 sets of experiments. Each set of experiment involves purification of 3 fragments of puc/taq I digest. The kit also includes electrophoresis equipment (ETS-1) required for agarose gel electrophoresis. KT43A : Kit is designed to carry out 5 sets of experiments. Each set of experiment involves purification of 3 fragments of puc/taq I digest. KT43B : Kit is designed to carry out 20 sets of experiments. Each set of experiment involves purification of 3 fragments of puc/taq I digest. Note : Electrophoresis equipment is required for KT43A and KT43B. 4 5
Duration of experiment: Experiment is carried out over a span of 2 days, approximate time taken on each day is indicated below: Day 1 : 4 hours (Agarose gel electrophoresis of puc/taq I digest and excision of individual bands) Day 2 : 5 hours (Extraction of DNA and analysis by agarose gel electrophoresis) 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 KT43/43A KT43B Store (5 sets of expts.) (20 sets of expts.) puc/taq I digest 50 µg 4 x 50 µg 4 C Silica 0.25 ml 1.0 ml 4 C Sodium iodide 15 ml 60 ml 4 C solution 1X TE 1 ml 2 ml 4 C 2.5X Gel loading 0.25 ml 2 x 0.25 ml 4 C buffer Wash buffer 10 ml 25 ml 4 C Agarose 2.5 g 10 g RT 50X TAE 20 ml 80 ml RT 6X Staining dye 40 ml 160 ml RT 1.5 ml vials 50 Nos. 4 x 50 Nos. RT Materials Required: Equipment : Dry bath, Rocker (optional), Table top centrifuge, Vortex Mixer. Glassware : Beaker, Conical flask, Measuring cylinder, Staining tray. Reagents : Distilled water, Isopropanol. Other Requirements : Gel cutter / blade, Micropipette, Thermometer, Tips, Water bath. 6 7
Note: Read the entire procedure before starting the experiment. Wear gloves while handling sodium iodide solution. Set the dry bath at 55 C prior to starting the experiment. In case a water bath is being used, ensure that the temperature is maintained at 55 C for effective solubilization of gel. Wipe the gel cutter or blade with Isopropanol after excision of each individual band of the digest from agarose gel, to avoid cross-contamination. puc/taq I digest supplied as ready to use is prepared by digesting plasmid DNA puc18/19 with Taq I restriction enzyme. This can be loaded directly onto agarose gel. As the experiment involves handling agarose gel, do not stain the gel with EtBr, use the staining dye provided. For preparation of agarose gel, staining etc., refer Agarose Gel Electrophoresis Procedure: Day 1: Electrophoresis of puc/taq I Digest. 1. Prepare a 1% agarose gel. 2. Load 25 µl (5 µg) of the digest on a 1% agarose gel. 3. Electrophorese the sample at 50-100 V till the blue dye has run 3/4 th of the distance. Switch off the power supply, disconnect the cords. Note : Pour out 1X TAE into a container, for later use. 4. Excise precisely with a sharp blade the lane in which the digest has been run. Take care not to dislodge rest of the gel or cut the other wells. 5. Cover the rest of the gel with 1X TAE and store at 4 C for later use. 8 6. Place the excised agarose block in a small tray and stain using 1X staining dye. Ensure that the gel is completely immersed in the dye. For uniform staining, place the tray on a rocker or shake intermittently every 10 to 15 minutes. 7. After an hour, pour out the staining dye into a container. Note : Store the staining dye at room temperature (RT) for later use. 8. Destain the gel using tap water till DNA is visible as a dark blue band against a light blue background. 9. Cut out each band of the digest precisely and place them separately in 3 different pre-weighed 1.5 ml vials. Mark them as 1, 2 and 3. Note : Store the vials at 4 C and continue with the experiment next day. Day 2: Extraction of DNA 10. Weigh each of the gel piece and add 2.5 volumes of sodium iodide solution. For example, if the gel weighs 0.1g add 250 µl of sodium iodide. 11. Solubilize the gel at 55 C by incubating in a water bath or dry bath for 5 minutes. 12. Tap the vial to mix the contents and incubate for another 5 minutes to ensure complete solubilization. 13. Vortex silica solution till it is uniform in suspension. 14. Add 15 µl of silica solution to each of the solubilized sample. Leave it at room temperature for 10-15 minutes with intermittent mixing for adsorption of DNA molecules to silica. 15. Spin at 8000 rpm for 1-2 minutes and discard the clear supernatant. 16. Add 200 µl of wash buffer and vortex it. 17. Spin at 8000 rpm for 1-2 minutes. Discard the clear supernatant. Repeat this washing step (steps 16 & 17) once more. 9
18. Remove carefully any traces of wash buffer after the second wash. Incubate the vial at 37 C (Dry bath) to ensure complete drying of the pellet. Note: Do not use a water bath. 19. To elute DNA, add 15 µl of 1X TE buffer to each of the pellet and mix by mild vortexing. Incubate at 55 C for 10 minutes. 20. Spin at 8000 rpm for 1-2 minutes and collect the respective supernatants in fresh vials marked 1, 2 and 3 21. A second elution with 15 µl of 1X TE buffer will ensure good recovery of DNA. Note: Alternatively, a single step elution using 30 µl of 1X TE buffer can be done and steps 19 & 20 followed. 22. Collect the supernatants in the respective vials taking care not to mix the elutants. 23. Spin at 8000 rpm for 2 minutes to remove traces of silica from the sample. 24. Pipette out carefully the supernatant into fresh vials. 25. Add 6 µl of gel loading buffer to each of the vials. 26. Load the individual samples along with puc/taq I digest (25 µl) in different wells of the gel made earlier. Note down the order in which the samples are loaded. Note: Use fresh tip to pipette puc/taq I digest to avoid any contamination. 27. Electrophorese the samples as mentioned earlier (using the stored 1X TAE buffer). 28. Stain using 1X dye prepared earlier, for about an hour. Destain using tap water, till bands are seen. Observation: Compare the intensity of individual fragments with the corresponding bands of the marker and estimate the approximate percent of DNA recovered as follows: DNA fragment 1444 bp fragment 736 bp fragment 476 bp fragment Recovery Interpretation: Any desired DNA fragment can be purified from a pool of DNA fragments by a simple combination of agarose gel electrophoresis and binding to silica. Recovery of DNA by this method of purification varies from 60-70%. However, higher sized fragment (1444 bp) is recovered in larger amounts when compared to lower sized fragments i.e. 476 / 736 bp. (Refer Fig 1). DNA thus obtained is pure, free from contaminants and can be used for many applications. 1 2 3 4 Lane 1 : puc18/ Taq I Digest Lane 2 : 1444 bp fragment Lane 3 : 736 bp fragment Lane 4 : 476 bp fragment Fig 1 : Purified puc/taq I fragments run on 1% agarose gel (Stained with EtBr). 10 11
Agarose Gel Electrophoresis 12 13
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 Figure: 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. 14 15
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. 16 17
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. 18 19
Ordering Information Product Size Cat # 1 Pack KT43 Teaching Kit (Consumables for 5 experiments & Elpho Kit (ETS 1)) 1 Pack KT43A Teaching Kit (Consumables 5 experiments) 1 Pack KT43B Teaching Kit (Consumables 20 experiments) Email: Sales: geneisales@sanmargroup.com Customer Support: geneitechsupport@sanmargroup.com 20