Name John Denver Ignacio

Section BSA3 AS2

Laboratory Schedule

1. Search and discuss examples of conventional and kit-based isolation of DNA and
RNA from plant tissue. Mention what kind of tissue is applicable. Write them in a
workflow process form. You can also mention what brand and type of kit you
searched.

Rapid plant DNA and RNA extraction protocol using a bench drill

Most plant DNA and RNA extraction protocols available make use of liquid
nitrogen as the main component to disrupt the cell wall and give access to these
molecules. However, liquid nitrogen can be expensive, hindering the extraction of
a large number of samples, mainly due to its availability and cost and depending
on each laboratory conditions and budget.
Although there are various published protocols that do not use liquid
nitrogen, our protocol makes use of a low cost bench drill to substitute the liquid
nitrogen to hasten extraction and cheap plastic bags instead of mortar and
pestle, yielding large quantities of high quality DNA and RNA from leaves and
roots of a variety of tropical plant species. This protocol can be used for
extraction of both DNA and RNA of plants, and while usually nucleic acid
extraction by other protocols makes use of mortar and pestle, this protocol
eliminates the differences in the amount and quality of nucleic acids extracted
providing homogeneity in samples since the same force (drill) is applied.
Furthermore, in samples with high water content and in woody samples,
maceration using mortar and pestle is hindered due to formation of ice crystals
whereas this problem is overcome by homogenizing samples using a drill. Also,
most RNA extraction protocols require expensive kits and laboratory equipment,
such as a refrigerated centrifuge, which is not necessary with the use of this
protocol. It also delivers good quality RNA that can maintained at room
temperature for at least three weeks.
The round shape of the Biorema drill (Agdia® ) facilitates homogenization
of the sample. It is made of an aluminum steel rod with a 4 cm diameter bottom,
with ten ball bearings of approximately 0.5 cm diameter each around its
circumference, attached to the drill bit. This protocol can be used in a laboratory
with minimal conditions and equipment, since it does not require the use of a
refrigerated centrifuge during the different steps, and is best recommended for
fresh samples that were not stored at -80°C, or were not subjected to ultra-low
temperatures during the collection procedure. Moreover, samples can be easily
stored in a simple freezer (- 18°C) for 3 - 4 days prior to nucleic acid extraction.
Due to the instability of the RNA molecule and sometimes the need for
transportation to other institutions (for high-throughput sequencing HTS -
analysis, for example), which may take days or weeks, the shelf life of RNA
pellets is a relevant concern.

Materials and Method:

DNA and RNA from leaves of banana, passion fruit, papaya, citrus,
pineapple and cassava, and RNA from roots of banana, citrus, pineapple and
cassava, were extracted using a bench drill. Young leaves and roots of most of
the tropical plants mentioned above were collected from the germplasm bank at
Embrapa Mandioca e Fruticultura located in Cruz das Almas, Bahia, Brazil.
DNA and RNA of three plant genotypes per species (with three replicates
per genotype), were extracted using the CTAB (cetyltrimethylammonium
bromide) protocol Genetics and Molecular Research 18 (3): gmr18394 ©FUNPEC-
RP www.funpecrp.com.br Inexpensive DNA and RNA extraction protocol 3 (Doyle
and Doyle, 1990) protocol and the CIP - CTAB protocols (see below) for DNA and
RNA extractions, respectively. In both cases, the first step of the protocol was
modified with the use of plastic bags (20 x 10 x 0.01 cm - virgin low-density
polyethylene - Bahia Embalagem® - or any other supplier that offers the same
specifications) and a drill to grind plant tissues inside the plastic bags at rotation
of 2500 rpm until tissue is fully dissolved. The drill bit can be cleaned with 70%
alcohol for each sample change. The drill at this rotation in contact with bags
with the specifications above, will not damage the bag, however, two bags
(inside each other) may be used if suited. Two mL of extraction buffer (2%
CTAB, 100 mM, Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0 . 1.4 M NaCl, 2% PVP-40
- add before use and 1% Na sulfite add before use) were added in plastic bags
containing 300 mg of leaf tissue and macerated by using Biorema drill (Agdia®).

FavorPrep TM Plant Genomic DNA Extraction Mini Kit

Genomic DNA Mini Kit provides a fast and simple method to isolate total DNA
(genomic DNA, mitochondrial and chloroplast) from plant tissue and cells. In the
process, sample is distrusted by grinding in liquid nitrogen and lysis buffer
incubation. The Lysate is treated with RNase A to degrade RNA and filtrated by
filter column to remove cell debris and salt precipitations. In the presence of
binding buffer with chaotropic salt, the genomic DNA in the lysate binds to glass
fiber matrix in the spin column. The contamin are washed with an ethanol
contained wash buffer and finally, the purified genomic DNA is eluted by low salt
elution buffer or water. The protocol does not require DNA phenol extraction and
alcohol precipitation. The entire procedure can be completed in 60 minutes. The
purified genomic DNA is ready for PCR, real-time PCR, Southern blotting and
RFLP.

Step 1: Tissue Dissociation

Cut off 50mg (up to 100mg) of fresh or frozen plant tissue or 5 mg (up to 100
mg) of dried sample. Grind the sample under liquid nitrogen to a fine powder.
Transfer it into a microcentrifuge tube (not provided). For some plant sample, we
can destruct it without liquid nitrogen.

Step 2: Lysis
Add 400 µl FAPG1 Buffer and 8µl RNase A vortexing. Do not mix FAPG1 Buffer
and RNase A before use. Incubate at 65 C for 10 minutes. During incubation,
invert the tube every 5 minutes. At the same time, preheat required Elution
Buffer (200µl per sample) at 65 C. Add 130µl FAPG2 Buffer and mix by vortexing.
Incubate at ice for 5 minutes. Place a Filter Column in a 2 ml Collection Tube.
Apply the mixture from previous step to the speed (13,000 rpm). Discard the
Filter Column and carefully transfer clarified supernatant in Collection Tube to a
new microcentrifuge tube (not provided).

Step 3: DNA Binding

Add 1.5 volumes of FAPG3 Buffer (ethanol by vortexing for 5 seconds. For
example, add 750µl Place a FAPG Column in a 2 ml Collection Tube. Apply 750µl
the mixture (including any precipitate) from previous step to the FAPG Column.
Centrifuge at full speed (13,000 rpm) for 2 minute. Discard flow-through in
Collection Tube and apply remaining mixture to FAPG Column. Centrifuge at full
speed (13,000 rpm) for 2 minute. Discard flow-through in Collection Tube.

Step 4: Wash
Add 500µl of W1 Buffer (ethanol added) into the column. Add 750µl of Wash
Buffer (ethanol added) into the column. Centrifuge at full speed (13,000 rpm) for
30 seconds. Discard the flow-through and place the FAPG Column back in the
Collection Tube. Centrifuge at full speed for 3 minutes to dry the column matrix.
---Important Step! The residual liquid can affect the quality of DNA and inhibit
subsequent enzymatic reactions.

Step 5: DNA Elution

Transfer dried FAPG Column into a clean 1.5 ml ‧Add 50-200µl of preheated
Elution Buffer into the ‧Stand for 3 minutes until Elution Buffer absorbed be used,
reduce the elution volume (50-150µl) to increase DNA concentration.
microcentrifuge tube (not provided). center of the column matrix. by the matrix.
‧Centrifuge full speed (13,000 rpm) for 2 minutes to elute purified DNA.
---Important Step! For effective elution, make sure that the elution solution is
dispensed on the membrane center and is absorbed completely. can affect the
quallity of DNA and inhibit subsequent enzymatic reactions.

RNA Extraction and Purification from Fresh or Frozen Plant Tissue

E.Z.N.A.® Plant RNA Kit

The E.Z.N.A.® RNA family of products is an innovative system that

radically simplifies the extraction and purification of RNA from a variety of
sources. The key to this system is that it uses the reversible binding properties of
the HiBind® matrix (a silica-based material) in combination with the speed of
mini column spin technology. Single or multiple samples can be processed
quickly. There is no need for phenol/chloroform extractions and time-consuming
steps, such as CsCl gradient ultra-centrifugation and precipitation with
isopropanol or LiCL, are eliminated.
RNA purified using the E.Z.N.A.® RNA purification system is ready for
applications such as RT-PCR, Northern blotting, poly A+ RNA (mRNA)
purification, nuclease protection, and in vitro translation.
The E.Z.N.A.® Plant RNA Kit can purify up to 100 µg plant RNA that is
>200 nt. Normally, 10-100 mg plant tissue can be processed in a single
experiment.
Lysis of cells or tissue occurs under denaturing conditions that inactivate
RNases. After the homogenization process, samples are transferred to the
HiBind® RNA Mini Column to bind RNA. Cellular debris and other contaminants
are removed after three quick wash steps. High-quality RNA is eluted in sterile
Nuclease-free Water

 Collect tissue in a 1.5 mL microcentrifuge tube (not provided) and freeze

by dipping in liquid nitrogen with a pair of tweezers to fill the tube. Grind
the tissue using disposable pestles. Do not allow samples to thaw.

 Transfer up to 100 mg frozen ground plant tissue to a new 1.5 mL

microcentrifuge tube. Samples should not be allowed to thaw before the
addition of RB Buffer in Step 3.

 Immediately add 500 μL RB Buffer mixed with 2-mercaptoethanol. Vortex

at maximum speed to mix thoroughly. Ensure that all the samples are
completely suspended and that there are no clumps in the solution.
Clumps will result in low yields.

 Insert a Homogenizer Mini Column into a 2 mL Collection Tube. Transfer

the lysate to the Homogenizer Mini Column. Centrifuge at
  14,000 x g for 5 minutes at room temperature. Discard the Homogenizer
Mini Column.

 Transfer cleared lysate to a new 1.5 mL microcentrifuge tube. Do not

disturb or transfer any of the pellet. Measure the volume of the lysate.

 Add 1 volume 70% ethanol. Vortex at maximum speed for 20 seconds. A

precipitate may form at this point; it will not interfere with DNA isolation.
Passing the mixture through a needle using a syringe or by pipetting up
and down may break up the precipitates.

 Insert a HiBind® RNA Mini Column into a 2 mL Collection Tube.

 Transfer 700 µL sample, including any precipitates that may have formed,
to the HiBind® RNA Mini Column. Centrifuge at 12,000 x g for 1 minute at
room temperature. Discard filtrate and reuse the collection tube.

 Repeat Step 8 until all of the sample has been transferred to the column.
 OPTIONAL: This the starting point of an optional on-membrane DNase I
Digestion protocol. If an RNA removal step is required, please continue to
the DNase I Digestion Protocol on the reverse page. (See DNase I
Digestion Set, Cat# E1091 for more information). If
 DNase I digestion is not required, proceed to Step 10.

 Add 500 μL RNA Wash Buffer I. Centrifuge at 10,000 x g for 30 seconds.

Discard the filtrate and the collection tube.

 Transfer the HiBind® RNA Mini Column to a new 2 mL Collection Tube.

2. Discuss briefly the differences between the conventional and the kit-based
protocols. Also, the differences between the DNA and RNA isolation from the
samples.

If you just need DNA for PCR, kit-based will be the best as it is fast and pure
from contaminants. But if you need more yield such as for a digestion,
hybridization you can go to conventional method. Here you can adjust the
sample amount and reagents volumes as you need. You cannot optimize any
reagents from kit to make what you want. But using conventional method, it can
be done.
 DNA Isolation

DNA can be prepared from a whole range of materials including blood, exfoliated
bladder cells, frozen tissue samples and paraffin tissue blocks. Since DNA is
generally stable under suitable storage conditions, it can be prepared and stored
in batches.

DNA isolation involves three basic steps – breaking the cells open to expose the
DNA, removing the membrane lipids through the use of an appropriate detergent
and precipitating the DNA with alcohol. There are also two optional steps that
are almost always done prior to the precipitation step - the removal of proteins
by adding an appropriate protease and the removal of RNA with the use of an
RNase.

Through DNA isolation of your samples, you can minimize the disturbance from
non-target DNA and other contaminants and increase the stability of your sample
in long-term storage.

RNA Isolation

On the other hand, it involves four basic steps. First, you need to disrupt the
cells by adding guanidium thiocyanate and a reducing agent to the sample and
then subject it to vigorous shaking or vortexing. This step will also break the
disulphide bonds and inactivate the contaminant proteins present in the sample.

After cell lysis, you need to add phenol and chloroform-isoamyl alcohol to
separate your RNA sample from the solution. The aqueous phase contains your
precious RNA so you need to put it in a separate tube, add isopropanol and
centrifugate the solution to precipitate your RNA. Washing the precipitate with
75% ethanol will then remove any impurities from your sample.

However, it is important to note that RNA is less stable as compared to DNA so

you should only isolate it prior to use. Special care should also be taken before,
during and after the process to ensure the purity of your sample. Remember,
even the smallest amount of RNase can compromise its quality.

3. What is the principle and purpose of DNA and RNA extraction?

 DNA extraction can be used to modify plants, by isolating DNA from organisms
with desirable traits, such as resistance to pesticides, and injecting them into the
genome of the plant. The basic principle of DNA isolation is disruption of the cell
wall, cell membrane, and nuclear membrane to release the highly intact DNA into
solution followed by precipitation of DNA and removal of the contaminating
biomolecules such as the proteins, polysaccharides, lipids, phenols, and other
secondary metabolites by enzymatic or chemical methods.

RNA extraction is the purification of RNA from biological samples. This procedure
is complicated by the ubiquitous presence of ribonuclease enzymes in cells and
tissues, which can rapidly degrade RNA. Isolation of intact RNA is essential for
many techniques used in gene expression analysis such as:
– Microarray analysis
– Northern analysis
– cDNA library construction
– RT-PCR

4. Differentiate genomic DNA, complementary DNA and plasmid DNA.

The complementary DNA is what you get from reverse transcription of RNA
(mRNA) while genomic DNA is the natural genetic material. In molecular cloning,
plasmids are types of vectors that are useful in cloning short segments of DNA.
The main difference is that genomic DNA has introns, cDNA doesn't.

But you cannot find cDNA in the cells (normally). Integration of plasmid means
the genomic DNA will be longer. You can easily check the length of genomic DNA
(and, thus, the success of transformation) with gel electrophoresis. Only need to
extract genomic DNA.

To go with cDNA, you would need to: assure yourself that your plasmid DNA is
being expressed, extract the cellular RNAs, do reverse transcription, check the
cDNA, amplify the sequence corresponding to plasmid DNA, sequence it and
compare with the original plasmid DNA.

5. Differentiate ribosomal RNA, transfer RNA and messenger RNA.

The messenger RNA carries genetic information from the nucleus to ribosomes
for the synthesis of proteins; while transfer RNA carries specific amino acids to
 the ribosomes to assist the protein biosynthesis, and on the other hand,
ribosomal RNA provides the structural framework for the formation of ribosomes.

6. Discuss the function and purpose of each chemical (Liquid Nitrogen, CTAB, SDS,
Chloroform, Ice-cold Isopropanol, Ethanol, TE Buffer, DNase/RNase Free Water)
used in CTAB Method.

Liquid Nitrogen
Liquid nitrogen is a chemical that is super cold, about -328°F (-200°C). Liquid
nitrogen will instantly freeze anything it touches. It is used to kill cells that make
up diseased or cancerous tissue. Tissue that has been frozen dries out and falls
off.

CTAB
The use of CTAB (cetyl trimethylammonium bromide), a cationic detergent,
facilitates the separation of polysaccharides during purification while additives,
such as polyvinylpyrrolidone, can aid in removing polyphenols. CTAB based
extraction buffers are widely used when purifying DNA from plant tissues.

SDS
Sodium Dodecyl Sulfate, Molecular Biology Grade (SDS), is a detergent that is
known to denature proteins. It is used in denaturing polyacrylamide gel
electrophoresis for the determination of protein molecular weight.

Chloroform
Chloroform is used as a solvent, a substance that helps other substances
dissolve. Chloroform increases the efficiency of phenol for denaturation of the
protein. Here, chloroform allows proper separation of the organic phase and
aqueous phase which keeps DNA protected into the aqueous phase. Chloroform
denatures the lipid as well.

Isopropanol
Isopropanol is used in precipitation of DNA, which breaks the hydration shell.
Isopropanol is a good choice for precipitation of DNA. The amount of isopropanol
requirement is less (0.6–0.7 volume of supernatant), as isopropanol has a higher
capacity to reduce the dielectric constant of water than the ethanol (2–3 volume)
and also requires a fair amount of salt to work. RNA which has extra 2′OH
remains hydrogen bounded with water more strongly than DNA tends to stay
soluble in it, thus selective precipitation of DNA can be done. Isopropanol also
dissolves nonpolar solvents such as chloroform, thus the impurities form previous
step can also be removed.

Ethanol
The main role of monovalent cations and ethanol is to eliminate the solvation
shell that surrounds the DNA, thus allowing the DNA to precipitate in pellet form.
Additionally, ethanol helps to promote DNA aggregation. Usually, about 70
percent of ethanol solution is used during the DNA washing steps.

TE Buffer
It dissolves DNA or RNA and protects the nucleic acid from degradation. It is a
major constituent of DNA extraction buffer which helps in lysis of cell wall and
nuclear membrane. It protects the nucleic acid from degrading by DNase or
RNase.

DNase/RNase Free Water

Nuclease-free Water is ideal for the preparation of reagents and for use in
enzymatic reactions. No toxic agents, such as DEPC, are used in the production
of this water, so as to avoid inhibition in enzymatic reactions.
References:
https://www.omegabiotek.com/product/e-z-n-a-plant-rna-kit/?cn-reloaded=1

https://www.interchim.fr/ft/E/EMV680.pdf

http://www.funpecrp.com.br/gmr/articles/year2019/vol18-3/pdf/gmr18394_-_rapid-
plant-dna-and-rna-extraction-protocol-using-bench-drill.pdf
https://microbenotes.com/rnaisolationprotocol/#:~:text=from%20Bacterial
%20Cel,Principle%20of%20RNA%20Isolation,GITC)%2C%20phenol%20and
%20chloroform.&text=Total%20RNA%20is%20then%20recovered%20by
%20precipitation%20with%20isopropanol.

https://byjus.com/biology/difference-between-plasmid-dna-and-chromosomal-dna/

https://medlineplus.gov/ency/article/002246.htm

https://opsdiagnostics.com/notes/protocols/ctab_protocol_for_plants.htm