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G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected]
A Geno Technology, Inc. (USA) brand name
think proteins! think G-Biosciences www.GBiosciences.com
PR033
Isolate Your Own Genomic DNA
Teacher’s Guidebook
(Cat. # BE-303)
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MATERIALS INCLUDED ....................................................................................................... 3
SPECIAL HANDLING INSTRUCTIONS ................................................................................... 3
ADDITIONAL EQUIPMENT REQUIRED ................................................................................ 3
TIME REQUIRED ................................................................................................................. 3
OBJECTIVES ........................................................................................................................ 4
BACKGROUND ................................................................................................................... 4
TEACHER’S PRE EXPERIMENT SET UP ................................................................................ 4
MATERIALS FOR EACH GROUP .......................................................................................... 5
PROCEDURE ....................................................................................................................... 5
PRECIPITATION OF GENOMIC DNA (OPTIONAL) ............................................................ 6
RESULTS, ANALYSIS & ASSESSMENT .................................................................................. 7
MATERIALS INCLUDED This kit has enough materials and reagents for 24 students (six groups of 4 students).
• 1 bottle Templating Buffer • 1 vial Protease: Dry Proteases • 1 bottle DNA Salt Solution • 2 bottles Precipitation Solution • 1 vial TE Buffer • 30 Cytology Brushes • 24 Centrifuge Tubes (2ml)
SPECIAL HANDLING INSTRUCTIONS • All reagents can be stored at room temperature
The majority of reagents and components supplied in the BioScience Excellence™ kits are non toxic and are safe to handle, however good laboratory procedures should be used at all times. This includes wearing lab coats, gloves and safety goggles.
For further details on reagents please review the Material Safety Data Sheets (MSDS).
The following items need to be used with particular caution.
Part # Name Hazard 344P Precipitation Solution Flammable
ADDITIONAL EQUIPMENT REQUIRED • 2 Waterbaths or beakers with thermometers • Pure or distilled water • 15ml Sterile tubes • Low Speed Centrifuge for 1.5-2ml tubes • Agarose gel electrophoresis equipment
TIME REQUIRED • Day 1: 2-3 hours
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OBJECTIVES • Isolate your own genomic DNA from your mouth • Study how to prepare genomic DNA for polymerase chain reaction (PCR),
restriction digestion and other molecular biology techniques.
BACKGROUND The isolation of genomic DNA is a highly important starting point for molecular biology. It allows researchers to obtain the starting material for subsequent cloning and genetic engineering. In addition, the isolation of genomic DNA is crucial in forensic science, where genomic DNA is routinely isolated from blood and cheek swabs. Genomic DNA can be isolated from numerous sources, including animal tissues, blood, cheek swab, plant, yeast, and bacteria.
This kit allows student to isolate their own genomic DNA that can be used for further applications.
TEACHER’S PRE EXPERIMENT SET UP 1. Prepare a waterbath or heating block at 50-60°C. Lower temperatures can be
used, down to room temperature, however longer digestion times will be required.
2. To visualize the genomic DNA, 1% agarose gels are required with enough wells for 1 per student. We recommend using the “Introduction to Agarose Electrophoresis” kit.
3. The purified genomic DNA can be used in conjunction with the “Polymerase Chain Reaction” kit to amplify a gene.
4. Prior to the commencement of the experiment, add 0.5ml Templating Buffer to the vial of dry protease to rehydrate. Mix by inverting the vial several times until a white suspension is visible. This solution can be stored frozen for later use.
Instruct students to stop eating and drinking at least one hour before experimentation. This helps prevent loose cells being washed away.
5. Prepare the DNA wash solution. Label a 15ml tube with “DNA Wash”. Transfer 10.5ml Precipitation Solution and 4.5ml pure or distilled water to the labeled tube. Invert 5-6 times to mix.
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MATERIALS FOR EACH GROUP • 3.5ml Templating Buffer • 4 Cytology brushes • 80μl Protease • 500μl DNA Salt Solution • 5ml Precipitation Solution • 2ml DNA Wash • 150μl TE Buffer • 5 Centrifuge Tubes (2ml)
PROCEDURE 1. Label a 2ml tube with your name.
2. Add 0.5ml Templating Buffer to a 2ml Tube. This solution contains detergents that disrupt the cell structure and releases the cell nuclei into the solution.
3. Collect Cheek Cells: Use the cytology brush to collect your cheek cells by scraping the inside of your cheek. Scrape the inside of each cheek; work on the area around the gum line 20 times, whilst twirling the brush between your fingers.
Instruct students not to scrape too vigorously. The best place for collecting the most cells is at the gum line.
4. Place the brush into the Templating Buffer and leave in the solution for 1 minute periodically twirling the brush.
5. Remove the brush from the solution, ensuring that you thoroughly scrape the brush on the side of the tube, releasing the cheek cells into the solution.
6. Vigorously shake, or vortex, the tube briefly for 30 seconds.
7. Pellet the nuclei: Centrifuge at high speed for 30 seconds. Remove and discard the supernatant.
8. Add 300μl Templating Buffer and resuspend the pellet by gentle pipetting. The Templating Buffer has detergents that release the genomic DNA from the nuclei.
9. Add 0.02ml Protease to the tube to digest the cellular material and release your genomic DNA into the solution.
10. Close the cap. Briefly mix by inverting the tube 10-15 times and then place in a 50-55°C waterbath or heating block for 1 hour.
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11. After 1 hour, transfer the tube to the boiling waterbath (>95°C) for 30 minutes to inactivate the protease enzyme, so it will not interfere with subsequent experiments.
CAUTION: Be careful around the boiling waterbath.
12. Store the genomic DNA at -20°C; the DNA can be stored for 2-3 months. Prior to use briefly centrifuge to pellet the debris. Use 1-5μl supernatant for PCR analysis.
13. The genomic DNA can be visualized on a 1% agarose gel, load ~20μl.
Precipitation of genomic DNA (Optional) 1. OPTIONAL: In addition, the genomic DNA can be precipitated. After the
centrifugation step in step 12 transfer the supernatant to a new tube.
2. Add 0.1ml of salt solution to reduce the solubility of your DNA and allow DNA precipitation in the next step.
3. To precipitate DNA from solution, an alcohol precipitation is carried out. Add 1.2ml DNA Precipitation Solution. Place your tubes on ice or at -20°C for ≥20 minutes.
4. Spin at maximum speed in a microcentrifuge for 5 minutes.
5. A small tight white pellet of DNA should be seen. Carefully decant the supernatant.
6. Add 0.5ml DNA Wash and swirl gently to mix.
7. Spin at maximum speed for 2 minutes.
8. Carefully decant supernatant and using a pipette remove all residual liquid.
9. Leave the tube open and allow the DNA to air dry. Once the pellet is completely dry add 30μl TE buffer and resuspend by gentle pipetting.
CAUTION: If the pellet is not allowed to dry completely then residual alcohol will effect subsequent reactions
10. The DNA can be run on an agarose gel to visualize the DNA or can be subjected to restriction digestion analysis followed by agarose electrophoresis for analysis of the plasmids. The precipitated DNA can also be used for PCR analysis and other molecular biology applications.
See pre experiment set up for information on agarose gel electrophoresis.
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RESULTS, ANALYSIS & ASSESSMENT This is dependent on how the genomic DNA isolation was confirmed or used. Design questions dependent on the choice of technique used.
Last saved: 10/24/2013 CMH
www.GBiosciences.com
Page 8 of 8
G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected]
A Geno Technology, Inc. (USA) brand name
think proteins! think G-Biosciences www.GBiosciences.com
PR034
IsolateYourOwnGenomicDNAStudent’sHandbook
(Cat.#BE‐303)
Page 2 of 8
OBJECTIVES ........................................................................................................................ 3
BACKGROUND ................................................................................................................... 3
MATERIALS FOR EACH GROUP .......................................................................................... 3
PROCEDURE ....................................................................................................................... 4
PRECIPITATION OF GENOMIC DNA (OPTIONAL) ............................................................ 5
RESULTS, ANALYSIS & ASSESSMENT .................................................................................. 6
OBJECTIVES• Isolate your own genomic DNA from your mouth • Study how to prepare genomic DNA for polymerase chain reaction (PCR),
restriction digestion and other molecular biology techniques.
BACKGROUNDThe isolation of genomic DNA is a highly important starting point for molecular biology. It allows researchers to obtain the starting material for subsequent cloning and genetic engineering. In addition, the isolation of genomic DNA is crucial in forensic science, where genomic DNA is routinely isolated from blood and cheek swabs. Genomic DNA can be isolated from numerous sources, including animal tissues, blood, cheek swab, plant, yeast, and bacteria.
This kit allows student to isolate their own genomic DNA that can be used for further applications.
MATERIALSFOREACHGROUP• 3.5ml Templating Buffer • 4 Cytology brushes • 80μl Protease • 500μl DNA Salt Solution • 5ml Precipitation Solution • 2ml DNA Wash • 150μl TE Buffer • 5 Centrifuge Tubes (2ml)
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PROCEDURE1. Label a 2ml tube with your name.
2. Add 0.5ml Templating Buffer to a 2ml Tube. This solution contains detergents that disrupt the cell structure and releases the cell nuclei into the solution.
3. Collect Cheek Cells: Use the cytology brush to collect your cheek cells by scraping the inside of your cheek. Scrape the inside of each cheek; work on the area around the gum line 20 times, whilst twirling the brush between your fingers.
Instruct students not to scrape too vigorously. The best place for collecting the most cells is at the gum line.
4. Place the brush into the Templating Buffer and leave in the solution for 1 minute periodically twirling the brush.
5. Remove the brush from the solution, ensuring that you thoroughly scrape the brush on the side of the tube, releasing the cheek cells into the solution.
6. Vigorously shake, or vortex, the tube briefly for 30 seconds.
7. Pellet the nuclei: Centrifuge at high speed for 30 seconds. Remove and discard the supernatant.
8. Add 300μl Templating Buffer and resuspend the pellet by gentle pipetting. The Templating Buffer has detergents that release the genomic DNA from the nuclei.
9. Add 0.02ml Protease to the tube to digest the cellular material and release your genomic DNA into the solution.
10. Close the cap. Briefly mix by inverting the tube 10‐15 times and then place in a 50‐55°C waterbath or heating block for 1 hour.
11. After 1 hour, transfer the tube to the boiling waterbath (>95°C) for 30 minutes to inactivate the protease enzyme, so it will not interfere with subsequent experiments.
CAUTION: Be careful around the boiling waterbath.
12. Store the genomic DNA at ‐20°C; the DNA can be stored for 2‐3 months. Prior to use briefly centrifuge to pellet the debris. Use 1‐5μl supernatant for PCR analysis.
13. The genomic DNA can be visualized on a 1% agarose gel, load ~20μl.
Page 4 of 8
Precipitation of genomic DNA (Optional) 1. OPTIONAL: In addition, the genomic DNA can be precipitated. After the
centrifugation step in step 12 transfer the supernatant to a new tube.
2. Add 0.1ml of salt solution to reduce the solubility of your DNA and allow DNA precipitation in the next step.
3. To precipitate DNA from solution, an alcohol precipitation is carried out. Add 1.2ml DNA Precipitation Solution. Place your tubes on ice or at ‐20°C for ≥20 minutes.
4. Spin at maximum speed in a microcentrifuge for 5 minutes.
5. A small tight white pellet of DNA should be seen. Carefully decant the supernatant.
6. Add 0.5ml DNA Wash and swirl gently to mix.
7. Spin at maximum speed for 2 minutes.
8. Carefully decant supernatant and using a pipette remove all residual liquid.
9. Leave the tube open and allow the DNA to air dry. Once the pellet is completely dry add 30μl TE buffer and resuspend by gentle pipetting.
CAUTION: If the pellet is not allowed to dry completely then residual alcohol will effect subsequent reactions
10. The DNA can be run on an agarose gel to visualize the DNA or can be subjected to restriction digestion analysis followed by agarose electrophoresis for analysis of the plasmids. The precipitated DNA can also be used for PCR analysis and other molecular biology applications.
See pre experiment set up for information on agarose gel electrophoresis.
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RESULTS,ANALYSIS&ASSESSMENTThis is dependent on how the genomic DNA isolation was confirmed or used. Your teacher will supply appropriate questions and discussion topics.
Last saved: 10/4/2012 CMH
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www.GBiosciences.com
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