Transcript DNA EXTRACTION - Michigan State University

DNA EXTRACTION
Protocol and notes
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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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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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Overview of DNA Extraction
Break down
the cell wall
and
membranes
Centrifuge to
separate the
solids from
the dissolved
DNA
Dissolve
DNA
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Precipitate
the DNA
using
isopropanol
Wash the
DNA pellet
with Ethanol
and dry
pellet
Centrifuge to
separate the
DNA from
the dissolved
salts and
sugars
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Protocol for Collecting the Plant Material
1.
Set up and label (according to species) TWO
eppendorf tubes for each seedling you will be
extracting DNA from.
2.
Add 600 µL of isopropanol to one tube in step 1 and
leave the other tube empty.
3.
Add 2.0 mL of extraction
buffer to the mortar.
4.
Cut a 2 inch portion of leaf
from each plant and grind
in the mortar with the pestle
until the solution is deep green.
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Transfer 1.5 mL of solution to empty tube.
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Protocol (continued)
5.
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)
6.
Remove 1 mL of the supernatant and transfer to
the tube containing 600 µL of isopropanol.
DISCARD the tube with the debris pellet!
A centrifuge
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Product is the
supernatant
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Protocol (continued)
7.
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”)
8.
Centrifuge samples at
high speed for 5 minutes
to pellet DNA. Remove
supernatant and save the
pellet (DNA is in the
pellet!).
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The pellet of DNA
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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 10-15 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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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
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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.
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Ladder
A B C D E
Barley(A), Corn (B), Oat (C), and Rice (D) are all
suitable
Wheat (E) may need to be re-extracted
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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
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Cereal Genomic DNA
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