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
Water bath
Vortex
Dewar
Microcentrifuge tubes (blue, pink & yellow)
Collection tubes (clear)
DNeasy Mini Spin Columns (clear)
QIAshredder Mini Spin Columns (lilac)
Mortar and Pestle
Micropipettes and tips
Ice bath
Thermometer
REAGENT/CHEMICALS:
Buffer AP1
Buffer AE
H2O
Buffer AP2
Buffer AW with Ethanol
Onion
Buffer AP3
RNase A
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) microcentrifuge 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 flowthrough. 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
15-25ºC and 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
Spatula
Microwave
P10 Micropipette and Tips
Microcentrifuge Tubes
100 mL Graduated Cylinder
Analytical Balance
Heat-Resistant Gloves
Erlenmeyer Flask
Electrophoreses Chamber
Power Supply
Gel Mold and Dams
Magnetic Stir Bar
REAGENT/CHEMICALS:
Agarose Gel Powder
Loading Dye
1 X TAE Buffer
DNA Ladder
Ethidium Bromide (10 mg/mL)*
*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 graduated cylinder. 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 lightbox. BE SURE TO WEAR EYE-PROTECTION.
SPECTROPHOTOMETER
EQUIPMENT/REAGENTS:
Quartz Cuvettes
Tap Water
HP Spectrophotometer
Micropipette and Tips
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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