Experiment #3 o The principle of DNA isolation is to extract

1st Video DNA from samples like human cells,

Special Issues on Plant Genomes which bacteria cell, and plant material.
includes an online interactive feature with
informational graphics animation and videos.
Explore how plant genome research is
contributing to our understanding of plant biology
and evolution and is leading to tangible benefits
for society. There are 3 basic steps in DNA extraction:
Dean DellaPenna, Michigan State University Step 1: Cell Lysis
o The past decade or so of plant genetic 1. Add lysis buffer to your samples and
research/plant genomic research has incubate at least 30ºC to release DNA and
been really focused on casting as wide a proteins.
net as possible over the plant Kingdom. o Breaking the cell membranes or cell walls
o That is to identify those bacteria algae, with lysis buffer.
simple plants mosses, etc that are key o General solution of lysis buffer contains:
points in the evolution of higher plants that tris-HCL, EDTA, EGTA, SDS,
is angiosperms, gymnosperms, the trees deoxycholate, tritonX, NP-40
and flowers that we have in our yard.
o This tremendous diversity that has
occurred through evolution can be traced
back and really identified through fully
sequenced genomes. You can see how
pathways have evolved, how pathways
have developed, and where the bits and
pieces for pathways have come from
Step 2: Removing Proteins
during evolution.
2. Remove proteins with protease or
Philip Benfey, Duke University
precipitate with ammonium acetate
o Having the whole genome allows us to
3. Centrifuge the samples to pull proteins
know what all the genes are, and what
down and use supernatant for the next
their different classes of genes are, and
step.
how many genes there are in each class.
Step 3: DNA Purification
o This can be useful from the standpoint of
4. Wash supernatant in isopropanol and
eliminating certain genes or making good
then in ethanol
guesses as to the function of other genes.
o DNA is insoluble in isopropanol and
o Another great use is that we can compare
ethanol. By centrifugation, a pellet of DNA
the genomes of one plant species to
will form.
another and the plants within a species so
5. Dry the pellet at 65ºC and add ultra pure
across a population and this is a very
water.
useful for understanding how genes are
o Now you have purified DNA solved in
causing different traits to occur within a
water.
population or between populations
6. Analyze your DNA with PCR and DNA gel
between species.
electrophoresis.
2nd Video
3rd Video
DNA Isolation
This right here jeans promoters pop Roblox is
plants and we could we could get DNA from any
one of these plants we don't need we need very
little leaf tissue or we could use a lot of leaf issue
in this case I'm going to use a lot of leaf tissue I'm
going to use an entire League carefully take this
into this little tool here Thursday little bit we'll bring
that up to the lab to extract DNA from it bring this
up to the lab to extract DNA from it all right now
they were back in the lab but it had our 2X C cap
buffer star pizza please And I don't really need to
do this I'm gonna add linear polyacrylamide
anyway one microlitre linear probably so much
linear polyacrylamide is particularly useful if you're
going to use very minute pieces of plant sure that
we get a pallet at the end of this DNA extraction
so get feel confident that we didn't lose our DNA
once that's all had to take our pastor I'm gonna just
ride right until it's homogeneous just need to be so
quick if you have trouble to keep your stuff in there
you have to work this the more you grind the more
DNA you'll get and if you want to if you really want
to get all the DNA that you can out of a tissue like
say it's precious you would want to freeze this
tissue in liquid nitrogen first then grind it becomes
very easy to get a really a powder plant material
that's good enough i used a lot of leaf so I have a
lot of genomic DNA for a number of different
cloning's close that up I'm going to take a Kim wipe
to wipe it off and say that some bubbles on one
side I just give you some real brief spin down so
that's on the bottom that too showed it to now
everything is on the bottom and super green is
again like i said I'm trying to get a lot of genomic
DNA I'm gonna eat this at 65 degrees for 10
minutes we go longer come back to you after the
10 minutes is over 10 minutes are up see time as
this dissociated the nuclear proteins on the DNA
equal volume of chloroform we don't want this to
get we don't want this to be real hot so might
wanna give it a moment to cool down so easy way
to let it cool down it's just open the lid let it sit there
for a moment I could feel by my hands it's not it's
not terribly hot because I'm using a fairly small
bottle definitely wanna work with chloroform in the
fume hood it's carcinogenic make you pass out
hell too much of it now we need to homogenize
this go back to the bench marginalized at shaking
so it looks like a Milky Milky substance green
substance for plant material centrifuge spin for one
minute to separate the phases gonna start you can
see that we've separated phases on the bottom
we got the green chlorophyll pigments and stuff in
the chloroform phase and then we have at the
interface we have a whole bunch of plant material
that you can see there we want to take that upper
aqueous phase put it in YouTube usually get a
little bit less liquid than you started with so
whatever whatever seat that you started with you
probably get a little bit we really don't want to take
any of the interface it's better to be it's better to
leave a little bit of stuff behind than it is to come he
created can you show that you so we have a pretty
clear liquid does have a little bit of greenish color
here isopropanol to it precipitate both the DNA and
that linear acrylamide we want to mix for genomic
DNA we like to mix by shaking gentle shaking not
by necessarily vortexing because it's so big that he
can actually cheer and separate a little bit break
up a little bit centrifuge I will give it a 5 minutes 5
minutes Max speed hi sorry spin is done or being
a is not precipitated on the bottom because I I
started with such a large amount of DNA our plant
material you can actually see a pellet there i'm not
sure that will show up on the camera but there is
a pallet there that will be of course due to linear
acrylamide and be acarefully by putting it away
now I'm gonna watch this one ethanol 70%
ethanol can you show that here having to it's not
easy to see that ballot it will become much easier
to see the fella once you have 70% ethanol in a lot
of cases you're not going to see any felon with
isopropanol because isopropanol makes it
telepherique fairly clear we have a small belletti to
look kind of like maybe visible are my Jelly if you're
Jelly and once we had 70% ethanol the fellow
becomes even more wide so we want to just mix
this few times God now you can see this Bell it put
it put it against the black background maybe you
can see that the pellet has actually now dislodged
from the wall there and see that so we're gonna
just give this a brief spin 2nd quick spin is not just
to make sure the pallet is toward the bottom of the
tube and attached again you can see there I'm
going to just go to the same South carefully make
sure Bella doesn't doesn't fly out see it I'm gonna
come to the centrifuge here give it 5 second spin o Ethidium bromide binds to DNA and is
just to get the ethanol to the bottom of the two man fluorescent when exposed by UV light.
we're going to get rid of that super heavy o Be careful with ethidium bromide as it is
This is what I have for spending I've had slowly I'm very toxic.
gonna type it on the opposite side of the tube there 6. Pour the gel into the tank
where the pellet is I bet it slowly so that ethanol
stays together and we don't have any droplets of
ethanol left here against droplets of ethanol left in
your tube it will take longer for this to drag and see
I don't have it really any any major ethanol droplets
I just put this on the bench on the side right here
like this or we could do this believing that Cooper
is the preferred way just turn out to rack well let it
dry for about a minute and then we'll add Tris 7. Place a comb
buffer RT buffer to it longer term storage DNA is
found dry enough you don't want overdrive you
overdrive genomic DNA it may not go back into
solution very easily try on your bench of course
you don't have a big risk of that I am out of money
tiny amount of ethanol won't even hurt most of the
downstream on the page Christine I just had it
teeny bopper select at that it was a big ballot so
it's gonna take a little bit for it to go back to solution
what I'm going to do is I'm going to put the vortex 8. Let it set for 30 min and your gel is ready.
on very low speed just moves for tax and it can
also mix it by flicking it yes for genomic DNA is
probably desirable with a big fellow like that to just
let it sit for five or 10 minutes and then repeat what
I did here just a very gentle board texting and a
little bit of cooking and your DNA should be back
in solutions thank you for watching
4th Video
DNA Gel Electrophoresis: Principle and How it
Works
DNA Gel Electrophoresis
o A method used in biochemistry to
separate mixed DNA fragments to
o The gel is like a complex matrix of pores.
estimate the size and/or isolate the
The fragments are migrating through
fragments.
these pores.
How to make 1% agarose gel
o DNA molecules are negatively charged,
1. Weigh 0.5-gram Agarose in a 250 mL
so they migrate from the negative
flask.
electrode to the positive electrode.
2. Add 50 mL of 0.5 x TBE and mix
3. Microwave till the agarose melts
4. Let it cool down to 50 C
5. Add 1 microliter of ethidium bromide
(10mg/mL) and mix
 o Because of the pore, short DNA
fragments migrate easily, and long DNA
fragments travels slower.
o Therefore, the DNA fragments can be
separated by length.
How to prepare the samples
A sample contains:
o 20 ng of DNA
o 2-4 microliter of PCR reaction is often
enough
o Autoclaved demi water
o Loading dye
1. Loading dye is mixed with the DNA
samples to visualize and assess the
speed of the fragments migrating through
the gel.
2. DNA samples are added to the agarose
gel.
3. After the electrophoresis, the gel s ready
for analysis.
4. Expose the gel with UV light and the DNA
bands becomes visible.
How to Analyze?
o The first sample is a DNA ladder. This is
applied to estimate the size of the
fragments
o The size of the DNA bands is known
beforehand
o Compare other DNA bands with ladder
DNA bands to estimate the fragment
length.

o The gel with EthBr enables you to look at

DNA and/or isolate it
o By applying an electric field, DNA
molecules will move through the gel.

5th Video
Agarose Gel Electrophoresis
o This video will take you through the 3. Add electrophoresis running buffer to the
process for loading and running DNA reservoirs at each end of the gel chamber
samples on an agarose gel. and keep adding buffer into the wells in
o You will be using a mini sub cell to the gel are covered by at least 2
perform agarose gel electrophoresis. millimeters of buffer
o The sub cell has an electrode wire running
across the bottom of each end. This
provides from electric current to pass
which separates the DNA fragments in the
samples.

1. First, align the gels so that the wells are

closest to the negative or black electrode. 4. Place your samples to be loaded in the
DNA is negatively charged and will move correct order according to the lanes they
from the negative electrode through the are assigned to be running. Check your
gel toward the positive or red electrode. lab protocol for this information

5. To obtain a DNA sample, slowly press the

plunger on your adjustable micropipette to
2. Place the Agarose gel into the gel the first or soft stop.
chamber. 6. Holding the plunger down, insert the tip of
the pipette into the micro tube with the
DNA sample. Place the tip close to the
bottom of the sample then slowly released
the plunger button.
 match the terminals on the power supply
red to red and black to black.

7. when loading the samples, keep the

pipette tip perpendicular to the role of
wells. This will reduce the risk of
accidentally puncturing the wells with the 11. Switch the power supply on then set the
tip. Lower the tip of the pipette until it correct constant voltage for running the
breaks the surface of the buffer and is samples. See your lab protocol for this
located just above or just inside the well. information.
Slowly apply pressure to the plunger
button observe as a sample fills the well
make certain you stop at the first stop on
the pipette paused then slowly removed
the pipette while keeping your thumb
down on the plunger.

12. If a timer is available on your power

13. Press the start button to begin the flow of
8. Once all the samples have been loaded,
avoid any bumping or movement of the
gel chamber. This might result in the
sample spilling into adjacent wells.
9. Place the lid on the gel chamber with the
terminals correctly position to the
matching electrodes on the gel chamber
black to black and red to red.
supply, set the clock for the proper time
for your run
current that will separate the DNA
fragments. At this point you should be
able to see bubbles coming from the wires
at each end of the gel box. Notice that the
positive or red electrode has fewer
bubbles than the negative or black
electrode.

10. Connect electrodes to the power supply

again making certain the electrodes

14. In a few minutes, you should see the
samples begin to migrate from the wells
into the gel. Notice as your DNA samples
run, the diaphragm moves from the
negative electrode towards the positive
electrode.
Experiment #3: Genomic DNA Extraction and
Quantification from the Young Leaves of a
Philippine Medicinal Plant and its
Characterization using Agarose Gel
Electrophoresis
I. INTRODUCTION
Nucleic acids are required for the storage
and expression of genetic information in living
systems. There are two types of nucleic acids:
deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA). In eukaryotic organisms, DNA is located in
the nucleus, mitochondria, and the chloroplast in
plants. In prokaryotes, DNA is usually double-
stranded, circular, and is found throughout the
cytoplasm. However, a few viruses may contain
single-stranded DNA molecules.
In any molecular biology-related
experiment concerning nucleic acids, genes, and
genomes, DNA isolation is a primary and critical
step especially in the molecular analysis of a
certain biological entity. Hindrances for a
successful DNA isolation include the presence of
contaminants such as secondary metabolites,
phenolic compounds, enzyme-inhibiting
polysaccharides, and even essential oils.
Inactivation of DNAse, an enzyme that degrades
DNA, may also be employed to maintain intact
DNA structure on your genomic sample. In plant
DNA extraction, one of the most important steps is
the breaking down of the cell wall and cell
membrane to allow access to nuclear material
whilst preserving its structural integrity.
The Philippine Department of Health
(DOH) endorsed and recognized ten (10)
medicinal plants which have been thoroughly
tested and have been clinically proven to have a
signature medicinal value in the relief and
treatment of various ailments or diseases. The ten
medicinal plants include: (1) ringworm bush or
“akapulko” (Cassia alata) for the treatment of
ringworm and skin infections, (2) bitter gourd or
“ampalaya” (Momordica charantia) for its known
treatment in diabetes, (3) garlic or “bawang”
(Allium sativum) for the reduction of cholesterol
and blood pressure to hypertensive patients, (4)
guava or “bayabas” (Psidium guajava) as
antiseptic for wound and oral infections, (5) 5-
leaved chaste tree or “lagundi” (Vitex negundo) for
conferring relief against cough and asthma, (6)
Chinese honey suckle or “niyog-niyogan”
(Quisqualis indica L.) for its effective elimination of
intestinal worms in pediatric patients, (7) Blumea
camphor or “sambong” (Blumea balsamifera) for
its diuretic properties that helps in the kidney stone
excretion, (8) Philippine tea or “tsaang gubat”
(Ehretia microphylla Lam. or Carmona retusa) for
its effective treatment in intestinal motility and oral
infections, (9) Pepper elder or “ulasimang bato”
and “pansit- pansitan” (Peperomia pellucida) for
its effectiveness against arthritis and gout in
geriatric patients, and (10) Peppermint or “yerba
buena” (Clinopodium douglasii) for its use as an Group Set 1 Momordica Charantia
effective analgesic. Group Set 2 Psidium guajava
Analyses of these medicinal plants at the Group Set 3 Peperomia pellucida
molecular level (DNA or gene) will help us deduce Group Set 4 Any Philippine
various pieces of information or knowledge about medicinal plant
the plant and its medicinal value. The method B. The instructors will provide the following:
presented here is based on the cetyltrimethyl o Micro-centrifuge tubes Mortar and pestle
ammonium bromide (CTAB) nucleic acid
extraction procedure that makes it possible to C. Reagents and Substances
extract high molecular weight DNA within a short
period of time using small amounts of plant tissues 2X CTAB buffer (stored at room temperature) 2%
and without the use of expensive equipment and w/v CTAB
tedious processes. Purity of DNA will be o 1.4 M sodium chloride
determined spectrophotometrically, and DNA o 100 mM Tris-Cl, pH 8.00
profiling will be done via agarose gel o 20 mM EDTA, pH 8.00
electrophoresis (AGE). o 2% polyvinylpyrrolidone (PVP)
II. OBJECTIVES OF THE EXPERIMENT
After this experiment, the student must be able: Beta-mercaptoethanol (2-ME)
1. to isolate high molecular weight plant
DNA that can be used in standard CTAB-NaCl solution
molecular biology procedures and o 10% CTAB
experiments; o 0.70 M NaCl
2. to identify the mode of action of each
reagent or chemical used in genomic CTAB precipitation solution (stored at room
plant DNA extraction; temperature)
3. to determine the purity of the isolated o 1% w/v CTAB, 10 mM EDTA, pH 8.00, 50
DNA spectrophotometrically; and mM Tris-Cl, pH 8.00
4. to profile and characterize the isolated o ethanol, 80% aqueous solution
DNA using agarose gel electrophoresis.
TE buffer
o 10 mM Tris, pH 8.00
o 1 mM EDTA

III. Materials and Reagents Absolute isopropanol

A. The students will bring/borrow the EDTA (0.5 M, pH 8.00)

following:
TAE (Tris-acetate EDTA) buffer (working
o Ziploc (at least 3) concentration: 0.5X)
o Ice Chest
o Young leaves of a medicinal plant (at Sample loading buffer
most 50 g)
o Complete PPE Chloroform-isoamyl alcohol, 24:1 v/v solution
Group Set Assigned Medicinal
Plant 10.0 M sodium acetate

SYBR Green solution in DMSO (10,000x
preferably)
D. Equipment
o Centrifuge machine
o Freezer
o Water bath
IV. Methodology
A. Isolation of Genomic Plant DNA from the
Young Leaves of a Philippine Medicinal Plant
1. Harvest young leaves, rinse water to
remove dirt, and dry completely. Store
young plant leaves overnight in -20°C or
if this is not available, a freezer prior to the
day of extraction.
2. On the day of extraction, remove leaf
midribs and stems from young leaves.
Place collected tissues in a pre-chilled
mortar and pestle or if possible, work
using an ice bath. Grind the plant tissue
into a fine powder and transfer 0.1 g - 0.2
g into a micro-centrifuge tube.
3. To the 2X CTAB buffer, add the
appropriate amount of β-ME to give the
solution a final concentration of 5% v/v.
This should be done only prior to use. Pre-
heat this solution and the CTAB-NaCl
solution to 65°C.
4. To the ground tissue, add 500 µL of the
warm β-ME-CTAB buffer. This mixture
should be gooey and viscous. If this
consistency is not yet attained, add more
of the β-ME/CTAB buffer.
5. Incubate the mixture for 10 minutes at
65°C with continuous swirling.
6. Add an equal volume of 24:1 chloroform-
isoamyl alcohol to the mixture and mix
well by inversion.
7. Centrifuge at 12000xg for 1 minute at
room temperature to separate the
phases. Carefully separate the top
aqueous phase and transfer to a new
tube.
8. Add 1:10 volume of warm CTAB/NaCl
solution to the recovered phase and mix
well by inversion.
9. Repeat steps 6 and 7.
10. Add 1.5 volumes of CTAB precipitation
solution to the mixture and mix well by
inversion.
11. Add half a volume of 10 M Potassium
acetate afterwards. Invert the tube
several times. Keep the mixture on ice for
5 minutes.
12. Centrifuge at 12000xg for 10 minutes at
4°C. Recover the supernatant and
transfer to a new micro- centrifuge tube.
Discard the precipitate.
13. Add an equal volume of ice-cold absolute
isopropanol to the supernatant. Incubate
for 20 minutes on ice.
14. Centrifuge at 12000xg for 10 minutes at
4°C. Recover the pellet and discard the
supernatant transfer to a new micro-
centrifuge tube. Discard the precipitate.
15. Wash the pellet with 80% EtOH.
16. Air-dry the pellet and resuspend in 100 µL
of TE buffer.
17. Properly label the micro-centrifuge tube
containing the redissolved DNA pellet and
store at -20°C.
B. Spectrophotometric Quantification of the
DNA Isolate
1. Dilute a DNA isolate to a total volume of
1.00 mL.
2. Read its absorbance at 260 nm and 280
nm. Record your results accordingly.
3. Calculate the absorbance ratio
A260/A280.
4. Take note that an absorbance ratio of
1.80 indicates that the DNA isolate is
pure, while at lower values, impurities
which can be proteins, polysaccharides,
and/or polyphenols are present.
5. Assuming that you are dealing with pure
double-stranded (ds) DNA, determine the
concentration of the DNA in solution.
Accepted Spectrophotometric Nucleic Acid
Conversion Values
1.0 A260 unit of ds 50 µg/mL
DNA
 1.0 A260 unit of ss 33 µg/mL
DNA
1.0 A260 Unit of RNA 40 µg/mL
C. Characterization of DNA using Agarose Gel
Electrophoresis
1. Prepare and cook 0.7% agarose gel (100
mL) using TAE buffer in a microwavable
flask.
2. Microwave for 1-5 minutes with 30-45
second-interval of stop and swirl until the
agarose is completely dissolved. Swirl
occasionally. Do not over boil the solution.
3. When it is already approximately 50-60
oC (approaching to a cooled state), add
1.00 µL stock SYBR Green solution (in
DMSO per 10 mL of the gel solution). Mix
well. Pour the gel onto the electrophoresis
setup (later in the gel box) to solidify
(around 20-30 minutes). Do not forget to
place the combs to shape the sample
wells.
4. Mix 14 µL sample to 6 µL of sample
(loading) buffer by repeated pipetting. For
samples with highly concentrated DNA,
dilution and adjustments to sample and
buffer volumes might be necessary to
prevent smearing.
5. Pour 1X TAE buffer to the gel box until the
gel is completely submerged onto the
poured solution.
6. Load the molecular weight ladders in the
first and last lanes of the gel followed by
the samples on the succeeding empty
lanes. Run the gel at 80-150 V until the
dye line is approximately 75-80% of the
way down the gel. Typical run time
proceeds approximately at 1-1.5 hours.
7. After the run, visualize your gels using a
gel documentation system.