DNA Isolation from an Onion
Todd Ottman, DeAnn Sabal, Kristi Thompson, Bincy Varghese, Renee Wilkins
January 22, 2007
Presented to: Dr. James Bidlack
Molecular Cell Physiology Lab
Method for DNA Isolation from a White Onion
OBJECTIVE:
The purpose of this experiment is to extract DNA from onion tissue. Then use a qualitative electrophoresis method to identify the presence of DNA as well as a quantitative spectrophotometer method to determine the percent yield of DNA obtained.
EQUIPMENT:
Microcentrifuge / Microcentrifuge tubes (blue, pink yellow) / Mortar and PestleWater bath / Collection tubes (clear) / Micropipettes and tips
Vortex / DNeasy Mini Spin Columns (clear) / Ice bath
Dewar / QIAshredder Mini Spin Columns (lilac) / Thermometer
REAGENT/CHEMICALS:
Buffer AP1 / Buffer AP2 / Buffer AP3Buffer AE / Buffer AW with Ethanol / RNase A
H2O / Onion / N2(l)
PROCEDURE:
*Note: the following procedure was taken from DNeasy Plant Mini and DNeasy Plant Maxi Handbook and modified to fit this specific lab.
***Latex gloves are recommended when dealing with DNA samples.
PRE-LAB PREPARATION:
1. Heat up water bath to 65ºC
2. Turn on Spectrophotometer
3. Preheat Buffer AE
4. Prepare ice bath
5. Chill mortar and pestle
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DNA EXTRACTION:
1. Freeze onion sample with liquid nitrogen, grind to a powder using a chilled mortar and pestle by striking or pounding the frozen sample with the pestle.
2. Weigh 0.5g of onion on an analytical scale.
3. Transfer the sample to a 2 mL centrifuge tube (blue).
4. Add 400 μL of Buffer AP1 and 4 μL of RNase A, then vortex vigorously. No tissue clumps should be visible. Continue to vortex or pipette to remove any clumps. Clumped tissue will yield lower DNA results.
5. Incubate the mixture for 10 minutes in a hot water bath at 65°C (approx. 149°F) and mix it 2-3 times during incubation by inverting the tube.
6. Add 130 μL of Buffer AP2 to the lysate, mix with pipette and incubate for 5 minutes on ice.
7. Place the sample in the microcentrifuge (NOTE: be sure to balance it!!!) and spin down the plant material/chunks until they form as much of a pellet as possible. Five minutes at 13200 rpm. (Our lab group believes this should be lower at 10000 rpm.)
8. Remove the liquid portion (the lysate) by pipetting and place it in the QIAshredder Mini Spin Column (lilac). Centrifuge for 2.5 minutes at 10000 rpm. (Our lab group believes this should be lower at 8000 rpm.)
9. Transfer by pipetting flow-through fraction from step 8 to a new (clear) micro-centrifuge tube. Some cellular material may have been transferred through and may have formed another smaller pellet in the collection tube, be sure not to disturb the cell-debris pellet.
10.Add 750 μL of Buffer AP3/E to the cleared lysate and mix by pipetting.
11. Apply 650 μL of the mixture from step 10, including a precipitate if any, which may have formed, to the DNeasy Mini Spin Column (clear). Save the remaining solution from step 10 for step 12. Centrifuge for 1 minute at 8000 rpm then discard the flow-through. Reuse the collection tube in step 12.
12. Repeat step 11 with the remaining sample from step 10. Discard flow-through and collection tube.
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13. Place DNeasy Mini Spin Column in a new clear 2 mL collection tube, add 500 μL Buffer AW to the DNeasy Mini Spin Column and centrifuge for 1 minute at 8000 rpm. Discard flow-through and reuse the collection tube in step 14.
14. Add 500 μL Buffer AW to the DNeasy Mini Spin Column and centrifuge for 2.5 minutes at 10000 rpm to dry the membrane.
15. Transfer the DNeasy Mini Spin Column to a 2 mL microcentrifuge tube (pink) with the lid removed, and pipette 100 μL of preheated (65°C) Buffer AE (yellow tube) directly onto the DNeasy membrane. Incubate for 5 minutes at room temperature 15-25°C and then centrifuge for 1 minute at 8000 rpm to elute.
16. Repeat step 15 once by pipetting an additional 100 µL preheated (65ºC) Buffer AE directly onto the same DNeasy membrane. Incubate for 5 minutes at room temperature 15-25ºCand then centrifuge for 1 minute at 8000 rpm to elute. (The elution from steps 15 and 16 will be together in the lower portion of the tube. This is the material that you will use to perform the remainder of this lab.)
Qualitative and Quantitative DNA Analysis Methods
ELECTROPHORESIS
EQUIPMENT:
Weighing paper / 100 mL Graduated Cylinder / Electrophoreses ChamberSpatula / Analytical Balance / Power Supply
Microwave / Heat-Resistant Gloves / Gel Mold and Dams
P10 Micropipette and Tips / Erlenmeyer Flask / Magnetic Stir Bar
Microcentrifuge Tubes
REAGENT/CHEMICALS:
Agarose Gel Powder / 1 X TAE Buffer / Ethidium Bromide (10 mg/mL)*Loading Dye / DNA Ladder
*CAUTION: Ethidium Bromide is a strong mutagen, be very careful when handling it or
any material containing it.
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PROCEDURE:
1. Prepare 400 mL 1X TAE buffer by adding 40 mL 10X TAE stock solution to 360 mL deionized H2O.
2. Weigh 1.0 grams of agarose powder per 100 mL of 1X TAE Buffer for a 1.0% agarose gel. In this case,a 100 mL solution will be made.
3. Measure 100 mL of 1X TAE buffer into a graduatedcylinder. Pour 50 mL 1X TAE from the graduated cylinder into a flask with a magnetic stir bar. Add agarose powder weighed in step 2 and swirl to mix. Add the remaining 50 mL to the flask and again swirl to mix.
4. Place the agarose solution in the microwave and heat at HIGH setting for 2 minutes. Check to see if the solution is completely clear (void of any whispy strands or floating particles). If still turbid, heat at 30 second intervals until the solution is clear. Do not let it boil. Total heating time should take no longer than 3 ½ to 4 minutes.
5. Remove the solution from the microwave and place on bench top to cool.
6. After the solution has cooled for approximately 5 minutes, add 10 L Ethidium Bromide (10 mg/mL) and swirl to mix.
7. Assemble the gel mold and carefully pour the solution into the center, allowing even distribution. Fill the gel mold half-way up and set aside until it is firm (this should take approximately 20 – 30 minutes). It will appear cloudy when fully polymerized.
8. Place the gel into the electrophoresis chamber. Pour 300 mL 1X TAE buffer, set aside earlier, into chamber to cover gel completely.
9. In a new centrifuge tube (blue) place 15 μL DNA and 5 μL loading dye and centrifuge for 30 seconds at 5000 rpm.
10. In another tube (yellow) repeat step 9 with 15 μL DNA ladder and 5 μL loading dye.
11. Diagram what sample and how much will be placed in each lane of your gel, avoiding the use of the two outer lanes.
12. Pipette the 20 μL samples into the wells, be careful to not overfill them or blow air with the pipette.
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13. Place the lid on the chamber and connect the wire from the side closest to the wells of the gel to the negative current and the other end to the positive. The DNA will migrate to the positive end.
14. On a setting of high, turn the electrophoresis chamber on and allow the loading dye, or dye front, to migrate approximately two-thirds of the way down the gel.
15. Carefully remove the gel and identify the DNA bands with the use of a UV light-box. BE SURE TO WEAR EYE-PROTECTION.
SPECTROPHOTOMETER
EQUIPMENT/REAGENTS:
Quartz Cuvettes / HP Spectrophotometer / Micropipette and TipsTap Water
PROCEDURE:
1. Locate Hewlett Packard Spectrophotometer and become acquainted with ancient software (ask Renee for assistance); recall that a spectrophotometer compares a blank cuvette to a sample cuvette; thus evaluating one variable, i.e., the purity of collected DNA.
2. Run “DNA” with acquisitions of 220 nm – 330 nm and then run “DNA scan.” Print results.
a. Blank: fill quartz cuvette to bend in cuvette with deionized water, and scan cuvette once in spectrophotometer.
b. Sample: fill quartz cuvette with 120 μL DNA and 1300 μL deionized water, invert cuvette to mix the DNA and deionized water, scan cuvette in both programs.
3. Perform the following calculations using your acquired data.
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Calculations
Measured Peaks Absorbance: A260 =
A280 =
Note Absorbance Ratio: A260/A280 =
0.0 < 1.8 = Protein contamination
1.8 to 1.9 = DNA
1.9 to 2.0 = RNA contamination
Calculations for the DNA Yield:
(1) µg DNA per mL:
Absorbance: A260
Dilution factor: final volume/volume of extract added
Conversion factor: 50 μg/mL
(Absorbance)(Dilution factor)(Conversion factor) = µg/mL DNA
(2) µg DNA:
(µg/mL DNA)(Total volume from steps 15 and 16 = 0.2 mL) = µg DNA
(3) μg DNA per g tissue: (mass of original onion tissue, 0.5 g)
(µg DNA/0.5 g) = μg DNA/g tissue
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