1. DNA EXTRACTION
Protocol and notes
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2. Introduction
This highlights a simple protocol for extracting DNA from cereal plant leaves
This protocol will produce a product that is suitable as a template for polymerase chain reaction (PCR) and restriction enzyme digestion
It will also contain RNA which can be removed with further purifications
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3. DNA Extraction Protocol Overview
Fresh plant material is collected
The plant material is mixed with extraction buffer and ground in a mortar and pestle
The resulting liquid is centrifuged to separate the solid plant material from the extraction buffer supernatant
The supernatant is mixed with ethanol to precipitate the DNA which will form a pellet at the bottom of the tube
The supernatant is discarded
The DNA pellet is washed with a diluted ethanol solution
The DNA pellet is dried and then dissolved in water or a buffer
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4. Overview of DNA Extraction
Break down the cell wall and membranes
Centrifuge to separate the solids from the dissolved DNA
Precipitate the DNA using isopropanol
Centrifuge to separate the DNA from the dissolved salts and sugars
Flowchart of the various steps
Dissolve DNA
Wash the DNA pellet with Ethanol and dry pellet
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5. Protocol for Collecting the Plant Material
Set up and label (according to species) TWO eppendorf tubes for each seedling you will be extracting DNA from.
Add 600 µL of isopropanol to one tube in step 1 and leave the other tube empty.
Add 2.0 mL of extraction
buffer to the mortar.
Cut a 2 inch portion of leaf
from each plant and grind
in the mortar with the pestle
until the solution is deep green.
Transfer 1.5 mL of solution to empty tube.
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6. Product is the supernatant
Protocol (continued)
Centrifuge the eppendorf tube with the extraction buffer + crushed leaf at high speed for 3 minutes (this will pellet the leaf debris at the bottom of the tube)
Remove 1 mL of the supernatant and transfer to the tube containing 600 µL of isopropanol. DISCARD the tube with the debris pellet!
Product is the supernatant
A centrifuge
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7. Protocol (continued)
Invert the sample tubes (with supernatant and isopropanol) 15 times and incubate them at room temperature for 2 minutes. (note: DNA should precipitate out of solution and be visible as “white and cottony”)
Centrifuge samples at high speed for 5 minutes to pellet DNA. Remove supernatant and save the pellet (DNA is in the pellet!).
The pellet of DNA
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8. Pipette tip being used to break up the DNA pellet
Protocol (continued)
9. Add 1000 µL of 70% ethanol to the DNA pellets. Mix by inversion 15 times. Centrifuge at high speed for 3 minutes. Remove and discard as much supernatant as possible. Again, save the pellet!
10. Leave the tube open and let it air dry for minutes. Add 50 µL of 1X TE to resuspend the DNA in solution.
Pipette tip being used to break up the DNA pellet
Some tips on resuspending the DNA pellet:
Use a pipette tip to break up the pellet to ease resuspension
Do not pipette DNA up & down repeatedly- this shears DNA
It is best to leave the DNA on ice overnight and let it slowly redissolve
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9. Practical Notes The DNA solution is now ready to use.
To keep DNA solution stable, keep it in the fridge for short term storage and in the freezer for long term storage.
After extractions, it is a good practice to run the DNA on an agarose gel to check its integrity and quality
Mix 10 µL of DNA with 10 µL of loading buffer
Load this mixture into a 1% agarose gel
Insert picture of Gel electrophoresis
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10. Examining the Quality of the DNA Extracted
The product of your DNA extraction will be used in subsequent experiments, poor quality DNA will not perform well in PCR or restrictive digests. You need to be able to assess the quality of your DNA extraction.
Note that genomic DNA has a very high bp, so one would expect a band at the top of the gel (near the well) if it was extracted correctly.
A B C D E
Ladder
Barley(A), Corn (B), Oat (C), and Rice (D) are all suitable
Wheat (E) may need to be re-extracted
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11. If the genomic DNA is extracted in a professional Molecular Biology laboratory, it will give clear distinct band in an electrophoresis gel
Note how the bands of genomic DNA are distinct and found in the very high bp range
Ladders Cereal Genomic DNA
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