HiPer Gel Extraction Teaching Kit (Column Based) Product Code: HTBM010 Number of experiments that can be performed: 10 Duration of Experiment Agarose Gel Electrophoresis: 1 hour Protocol: 1 hour Agarose Gel Electrophoresis: 1 hour Storage Instructions: The kit is stable for 6 months from the date of receipt Store Control DNA and DNA Sample at -20 o C Store 6X Gel Loading Buffer at 2-8 o C Other kit contents can be stored at room temperature (15-25 o C) 1
Index Sr. No. Contents Page No. 1 Aim 3 2 Introduction 3 3 Principle 3 4 Kit Contents 3 5 Materials Required But Not Provided 4 6 Storage 4 7 Important Instructions 4 8 Procedure 4 9 Agarose Gel Electrophoresis 6 10 Quantitation of DNA 6 11 Flowchart 7 12 Observation and Result 8 13 Interpretation 8 14 Troubleshooting Guide 9 2
Aim: To learn the technique of DNA purification from Agarose gel using column based method. Introduction: HiPer Gel Extraction Teaching Kit (Column Based) has many advantages over the crude methods of extracting DNA. It is fast, simple and does not contain harmful organic compounds such as phenol and chloroform. The DNA purification procedure using the miniprep spin columns comprises of three steps: Adsorption of DNA to the membrane Removal of residual contaminants Elution of pure genomic DNA HiElute Miniprep Spin column format allows rapid processing of multiple samples. The columns have a high binding capacity and high quality DNA is obtained from various species. Principle: HiPer Gel Extraction Teaching Kit (Column Based) simplifies isolation of DNA from standard agarose gel by the use of silica binding with a microspin format that eliminates the need for expensive resins, alcohol precipitation and hazardous organic compounds such as phenol and chloroform. DNA binds specifically to the advanced silica-gel membrane while contaminants pass through. PCR inhibitors such as divalent cations and proteins are completely removed in wash step, leaving pure nucleic acid to be eluted in the Elution buffer. The purified DNA can be used for further downstream applications. Kit Contents: The Kit can be used to perform extraction of DNA from standard agarose using membrane column. Table 1: Enlists the materials provided in this kit with their quantity and recommended storage Sr. Quantity No. Product Code Materials Provided Storage 10 expts 1 TKC015 Control DNA 0.11 ml -20 o C 2 TKC128 DNA Sample 0.11 ml -20 o C 3 DS0023 Gel Bind Buffer 14.5 ml RT 4 MB063 Isopropanol 5 ml RT 5 DS0030 Gel Wash Buffer 4.5 ml RT 6 DS0040 Elution Buffer 0.6 ml RT 7 DBCA02 HiElute TM Miniprep Spin Column (in PW1139 11 Nos. RT Collection Tube) 8 PW1139 Collection Tubes, Polypropylene (2.0 ml) 22 Nos. RT 9 MB002 Agarose 9.6 g RT 10 ML016 50X TAE 120 ml RT 11 ML015 6X Gel Loading Buffer 0.06 ml 2-8 o C 3
Materials Required But Not Provided: Glass wares: Conical flask, Measuring cylinder, Beaker Reagents: Distilled water, Ethidium bromide (10 mg/ml) Other requirements: U.V. Spectrophotometer, Tabletop microcentrifuge (with rotor for 2.0 ml tubes), Electrophoresis apparatus, Incubator, UVTransilluminator, Heating block or Water bath, Vortex mixer, Clean razor blade, spatula, Micropipettes, Tips Storage: HiPer Gel Extraction Teaching Kit (Column Based) is stable for 6 months from the date of receipt without showing any reduction in performance. On receipt, store control DNA and DNA sample at -20 o C and 6X Gel Loading Buffer at 2-8 o C. Other kit contents can be stored at room temperature (15-25 o C). Important Instructions: 1. Read the entire procedure carefully before starting the experiment. 2. Preheat the heating block or water bath to 55 o C. 3. Only up to 400 mg of agarose can be processed per column. 4. Ensure that clean & dry eppendorf tubes, tips are used for the procedure. 5. In gel extraction procedure since agarose gel electrophoresis has to be carried out twice, 1X TAE buffer can be reused. 6. Preparation of Gel Wash Buffer for 1 expt: To prepare 0.75 ml of Gel Wash Buffer for 1 expt, add 0.375 ml of Concentrated Wash Buffer to 0.375 ml of Ethanol (96-100%). Procedure: Read the important instructions before starting the experiment. A. Electrophoresis of DNA to be purified: 1. Prepare 0.8% of standard agarose gel (as given below in Agarose Gel Electrophoresis). Allow the solution to cool down to about 55-60 o C. Add 0.5 µl of Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature. 2. To prepare samples for electrophoresis, add 2 µl of 6X gel loading buffer for every 10 µl of DNA sample. Mix and load the sample into the well. 3. Electrophorese at 100-120 volts until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized. Note: Store the 1X TAE tank buffer for later use. 4. Excise the DNA bands from the Ethidium Bromide stained gel using a clean razor blade or scalpel blade using 312 nm UV light and store it in a pre-weighed plastic micro centrifuge tube at 4 o C. 4
Note: Cut above and below each fragment of interest minimizing the amount of agarose in the slice. Also minimize the amount of time the DNA is exposed to UV by having sterile pre-weighed labeled tubes ready for the slices to avoid degradation of DNA. B. Gel Extraction: 1. Weigh the micro centrifuge tubes again after adding the gel slice into it. Determine the approximate weight of the gel slice and accordingly add 3 volumes of Gel Bind Buffer per gel slice volume. Incubate at 55 o C for10 minutes with intermittent mixing every 2-3 minutes so that the agarose dissolves completely (the yellow colour of Gel Bind Buffer signifies a ph of < 7.5). For e.g. 100 mg of agarose gel slice requires 300 µl of Gel Bind Buffer. NOTE: Monitor the ph of the Gel: Gel Binding Buffer mixture after the gel has completely dissolved. If the color of the mixture turns violet, add 5 µl of 5M Sodium acetate (ph 5.2) to bring down the ph. After this adjustment, the color of the Gel: Gel Binding Buffer mixture should be light yellow. The agarose gel slice should be solubilized completely. 2. Add 1 gel volume of isopropanol to the sample and mix. For example, if the agarose gel slice is 100 mg, add 100 µl of isopropanol. 3. Load sample onto the column Load the sample onto the HiElute Miniprep Spin column, and centrifuge at 13,000 rpm for 1 minute. NOTE: The maximum binding capacity of the column is 700 µl. For sample volumes exceeding 700 µl repeat the column step for the remaining sample. 4. Discard the flow through and place the HiElute Miniprep Spin column in a new 2.0 ml collection tube. 5. Wash Add 750 µl of the Gel Wash Buffer to the HiElute Miniprep Spin column placed in a new 2.0 collection tube and centrifuge for 1 minute at 13,000 rpm. 6. Discard the flow through and place the HiElute Miniprep Spin column in the same collection tube. Spin for an additional 1 minute at 13,000 rpm. 7. Carefully transfer the HiElute Miniprep Spin column to a clean 2.0 ml collection tube provided. 8. Elution Add 50 µl of Elution Buffer directly onto the centre of the HiElute Miniprep Spin column. Incubate at room temperature (15-25 o C) for 1 minute. NOTE: Incubation at room temperature (15-25 o C) for 1 minute increases the elution efficiency. 9. Centrifuge at 13,000 rpm for 1 minute to elute DNA. NOTE: All centrifugation steps are carried at room temperature (15-25 o C). For short-term storage (24-48 hours) of the DNA, 2-8 o C is recommended. For long-term storage, 20 o C or lower temperature (-80 o C) is recommended. Avoid repeated freezing and thawing of the sample which may cause denaturing of DNA. The Elution Buffer will help to stabilize the DNA at these temperatures. 10. Quantitate the concentration of the DNA using a spectrophotometer or estimate the concentration by comparing its intensity with that of a DNA ladder of known concentration. 5
Agarose Gel Electrophoresis: Preparation of 1X TAE: To prepare 500 ml of 1X TAE buffer, add 10 ml of 50X TAE Buffer to 490 ml of sterile distilled water*. Mix well before use. Preparation of agarose gel: To prepare 50 ml of 0.8% agarose gel, add 0.4 g agarose in 50 ml 1X TAE buffer in a glass beaker or flask. Heat the mixture on a microwave or hot plate, swirling the glass beaker/ flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure). Allow solution to cool to about 55-60 o C. Add 0.5µl Ethidium bromide, mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature. Loading of the DNA samples: To prepare sample for electrophoresis, add 2 µl of 6X gel loading buffer to 10 µl of DNA sample. Mix well by pipetting and load the sample into the well. Load the Control DNA after extracting the DNA sample. Electrophoresis: Connect the power cord to the electrophoretic power supply according to the conventions: Red-Anode and Black- Cathode. Electrophorese at 100-120 volts and 90 ma until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized. * Molecular biology grade water is recommended (Product code: ML024). Quantitation of DNA: Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and the purity of the genomic DNA. Use Elution Buffer to dilute samples and to calibrate the spectrophotometer, measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz microcuvette. Absorbance readings at 260 nm should fall between 0.1 and 1.0. The 320 nm absorbance is used to correct background absorbance. Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. Concentration of DNA sample (µg/ml) = 50 x A 260 x dilution factor 6
Flowchart: Agarose gel electrophoresis Electrophorese the DNA samples on standard agarose 7
Excise the DNA band of interest from the gel Weigh the gel slice containing the DNA band Add 3 volumes of Gel Bind Buffer per gel slice volume Incubate at 55 o C for 10 minutes with intermittent mixing Add 1 gel volume of isopropanol to the sample and mix Load the entire contents onto the column Centrifuge at 13,000 rpm for 1 minute and discard the flow through Add 750 µl of Gel Wash Buffer and centrifuge at 13,000 rpm for 1 minute, discard flow through Centrifuge the column for an additional 1 minute at 13,000 rpm Add 50 µl of Elution Buffer Incubate at room temperature for 1 minute Centrifuge for 1 minute at 13,000 rpm to elute the DNA Pure DNA Observation and Result: 1 2 8
Extracted DNA Fig 1: Gel picture of extracted DNA from agarose gel Lane 1: Control DNA Lane 2: Extracted DNA Table 2: Absorbance of the extracted genomic DNA at 260 nm and 280 nm Sample Dilution Factor A 260 A 280 A 260 /A 280 Concentration (µg/ml) 1 2 3 Calculate the concentration of isolated DNA using following formula: Concentration of DNA sample (µg/ml) = 50 x A 260 x dilution factor. Interpretation: Spectrophotometric analysis and agarose gel electrophoresis will reveal the concentration and the purity of the genomic DNA. Use Elution Buffer to dilute samples and to calibrate the spectrophotometer, measure the absorbance at 260 nm, 280 nm, and 320 nm using a quartz microcuvette. Absorbance readings at 260 nm should fall between 0.1 and 1.0. The 320 nm absorbance is used to correct background absorbance. Purity is determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. The concentration of DNA is calculated by the following formula: Concentration of DNA sample (µg/ml) = 50 x A 260 x dilution factor Troubleshooting Guide: 9
Sr.No Problem Possible Cause Solution Gel slice was not completely melted After addition of Gel Bind Buffer to the gel slice, vortex the tube every 3-4 minutes during the incubation (at 55 o C) to dissolve the gel Gel slice was too large Only up to 400 mg agarose can be processed per column. 1 Very less amount of DNA or no DNA obtained Elution Buffer was not loaded directly onto the centre of the HiElute Miniprep Spin column Carefully load the Elution Buffer onto the centre of the HiElute Miniprep Spin column Binding of DNA to the column was inefficient Binding of the DNA is dependent on both ph and salt concentration. Ensure that an appropriate amount of Gel Bind Buffer was used for the weight of the gel slice The solutions may have precipitated. If any solution forms a precipitate warm at 55-65 o C until the precipitate dissolves completely. Allow it to cool to room temperature (15-25 o C) before use 2 Incomplete dissolution of DNA Traces of ethanol left in the tube Wash Buffer has to be completely removed or else traces of ethanol remaining in the tube may interfere with the enzymatic reactions. Ensure that the column is spin for an additional 1 minute during the wash step, in order to completely remove traces of Wash Buffer Technical Assistance: At HiMedia we pride ourselves on the quality and availability of our technical support. For any kind of technical assistance mail at mb@himedialabs.com PIHTBM010_O/0514 HTBM010-04 10