Department of Biochemistry and Molecular Biology

University of Dhaka

MS Practical

Isolation of Rice DNA by Miniprep Protocol

Submitted by

Md. Atai rabby

MS

Priciple

DNA extraction is an important step in molecular assays and plays a vital role in obtaining

highresolution results in gel-based systems, particularly in the case of cereals with high content of

interfering components in the early steps of DNA extraction.This is a rapid miniprep DNA

extraction method, optimized for rice, which was achieved via creating some modifications in

present DNA extraction methods, especially in first step of breaking down and lyses of cell wall,

and the use of cheap and frequent chemicals, found in every lab, in the next steps. The normal

quality and quantity was obtained by the method. The PCR based assays also revealed the

efficiency of the method.

The advantages of this method are: 1- it is applicable with both dry and fresh samples, 2- no need

to large weight samples, 3- no need to liquid nitrogen and 4- easy, rapid and applicable in every

laboratory.

Introduction

High-quality DNA is required for molecular biological studies of plants. Several DNA extraction

procedures for isolating genomic DNA from various plant sources have been described, including

the salt extraction method and the cetyltrimethyl ammonium bromide (CTAB) method and its

modifications . The need for a rapid and simple procedure is urgent, especially when hundreds of

samples need to be analyzed .

In most protocols the use of liquid nitrogen or freeze -drying (lyophilization) of tissue for the

initial grinding is necessary, and these processes are unavailable in many regions of the

world. After grinding the tissues in various extraction buffers, DNA is extracted with phenol-

chloroform, or the extract is dialyzed against EDTA and a buffered Tris-HCl solution . After

extraction, the aqueous phase is concentrated, either by ethanol or isopropanol precipitation or

with microconcentrators (e.g., the Wizard genomic DNA purification system; Promega,

USA). However, these methods are not time efficient for consistently obtaining PCR-quality

DNA from cereal plants, since they require that the tissues be ground in liquid nitrogen, followed

by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc.

However, in the protocols provided by Kang et al. (2004) the DNA concentrations from cereal

cops (rice and maize) were relatively low. They suggested that this may be because

homogenization using a hand-operated homogenizer with a plastic tip is incomplete, since the

leaves of these plants are stronger than the leaves of tobacco, potato, cabbage, lettuce, and Siberian

ginseng. For our purposes, we desire a simple and fast procedure for obtaining plant genomic DNA

for PCR, and good-quality DNA for complete enzyme digestion. Therefore, we present a protocol

for extracting genomic DNA from young or old, fresh or dry cereal plant leaves that is applicable

to a variety of organisms, regardless of the complexity of their genomes. In addition, we present a

rapid and reliable procedure for extracting genomic DNA for PCR from a small amount (~0.5

cm2) of leaf tissue.

MATERIALS and REAGENTS:

1. Morter and pestle 11. 2-Mercaptoethanol (2-ME)

2. Sterile forceps 12. CTAB-extraction solution

3. Spatula 13. CTAB-NaCI solution

4.Screw capped tubes 14. CTAB-precipitation solution

5. Eppendorf tubes 15. 24:1 (v/v) Chloroform-Isoamyl alcohol

6. Water bath 16. High salt TE buffer

7. Centrifuge machine 17. Isopropanol (Ice cold)

8. Pipettes 18. 80% Ethanol (ice cold)

9. Micro pipette

10. Agarose gel (0.8%)

EXPERIMENTAL STEPS:

1. On the first day four reagents were made. These were: TE buffer, CTAB- extraction solution,

CTAB-NaCI solution, CTAB- precipitation solution.

2. 0.2% (v/v) of 2-ME(50iiL) to CTAB-extraction solution(25mL) was mixed and preheated at 65

2C in a screw-capped tube.

3. 1gm of rice leaves was taken on a mortar and then liquid nitrogen (2/3 times)

was added to pest leaves as powder. and 6mL of CTAB+2-ME solution was added. Then it was

taken in a screw-capped tube and incubated for 30min at 65 °C with occasional mixing.

4. Equal volume of Chloroform : lsoamyl alcohol (24:1) was added and mixed well.

Then it was centrifuged at 4000rpm for 20 min and the upper layer (5 ml) was collected

into another screw capped tube.

5. 1/10 volume of preheated (at 65 °C) CTAB-NaCl solution was added (i.e. 1 ml) into the

recovered upper layer and mixed well by inversion.

6. Equal volume of Chloroform : Isoamyl alcohol (24:1) was added and mixed well.

7. Then it was centrifuged at 4000rpm for 20 min (2-3 times) and the upper layer

(4.5 ml) was collected in another screw capped tube.

8. Exactly equal volume of CTAB- precipitation solution was added and mixed well by inversion.

9. As precipitation was not visible, the mixture was incubated for 30min at 65 2C

10. The mixture was centrifuged at 4000rpm for 10 min.

11. The mixture was equally divided into two screw capped tubes (that means each

tube got 4.5 ml mixture).

12. 0.8 volume (i.e. 3.6 ml in each tube) of isopropanol was added, mixed well and

incubated the mixture in ice for 10 min.

13. Then the mixture was centrifuged at 4000rpm for 20 min and again the supernatant was

discarded.

14. The pellet was washed with ice cold 80% ethanol (2 times) and centrifuged at

4000rpm for 20 min.

15. then the pellet was dried and resuspended in 150 µL (i.e. 75 µl in each tube) of

TE buffer (100-200 µL) and the sample was stored in an eppendorf tube.

16. The DNA preparation was analyzed by using 0.8% agarose gel.

Agarose Gel Electrophoresis of DNA

The equipment and supplies necessary for conducting agarose gel electrophoresis:

• An electrophoresis chamber and power supply

• Gel casting trays, which are available in a variety of sizes and composed of UV-

transparent plastic.

• Sample combs, around which molten agarose is poured to form sample wells in

the gel.

• Electrophoresis buffer, usually Tris-acetate-EDTA (TAE)

Protocol of 50X TAE(for 500mL):

Tris base =121gm

Glacial acetic acid =28.56mL

0.5M EDTA =50mL

dH2O =300mL

Then the mixture was shacked by magnetic stirrer until dissolved. Then the solution was poured

into a measuring flask to fix up to the mark. Finally the solution was autoclaved.

• Loading buffer

• Tracking dye (DNA dye)

• 1X TAE buffer, was made by mixing 490mL dH2O and 10mL 50X TAE.

• Ethidium bromide

• dH2O

Procedure of (0.8%) agarose gel electrophoresis:

1. 0.8g of agarose powder was mixed with 2mL of electrophoresis buffer (50X TAE)

and 110mL dH2O in a conical flask.

2. Then it was heated in a microwave oven until completely melted.

3. After cooling the solution to about 60 °C, it was poured into a casting tray

containing a sample comb and allowed to solidify at room temperature.

4. After the gel had solidified, the comb was removed, using care not to rip the

bottom of the wells. The gel, still in its plastic tray, was inserted horizontally into the

electrophoresis chamber and just covered with 1X TAE buffer.

5. Then our sample was diluted at different dilution with dH2O and 5mL of DNA

dye (loading buffer) was added in each tube.

6. 10mL of the samples with different concentration were pipeted into the sample wells.

7. Then the lid and power leads were placed on the apparatus, and a current (100V) was applied.

Current flow was confirmed by observing bubbles coming off the electrodes. DNA will migrate

towards the positive electrode.

8. The distance of DNA migration in the gel was judged by visually monitoring

migration of the tracking dyes.

9. When adequate migration had occured, the gel was stained after electrophoresis

by soaking in a dilute solution of ethidium bromide and destained with dH2O.

10. Then the gel was placed on a ultraviolet transilluminator to visualize DNA concentration. and

photography was taken place shortly after cessation of electrophoresis.

OBSERVATION:

DNA concentration was seen in under uv light but DNA band was not found in gel elactrophoresis

possibly for very low amount of DNA.

DISCUSSION:

The main purpose of this experiment is the successful isolation of DNA from plant source.

Reagents used for this in different step are for different reasons. All are outlined below:

Tris: It is widely used as a component of buffer solutions, such as in TAE and TBE

buffers used in biochemistry, with an effective pH range between 7.0 and 9.2. It acts as a

buffer. Tris is an effective buffer for slightly basic solutions, which keeps DNA

deprotonated and soluble in water.

Chloroform & Isoamyl alcohol: More organic Chloroform removes lipids, denatures

more proteins & mixes less with aqueous phase Iaeding to more efficient extraction. IAA

creates hydrophobic environment to help the isolation procedure.

Liquid Nitrogen: It inhibits nuclease activity & also creates very cold condition to inactivate

enzymes. It is used to crush leaves by removing H20.

CTAB: "Cetyl Trimethyl Ammonium Bromide" is a cationic detergent whose main

purpose is to break plant cell wall & helps to bring out DNA easily preventing other

conventional method complications. All CTAB solution acts as a lysis solution.

2-ME: 2-Mercaptoethanol is used for reducing protein disulfide bonds to

precipitate protein which can be removed by centrifugation.

EDTA: EDTA chelates Ca+2 & Mg+2. So, nuclease action is inhibited. These ions are

necessary co-factors for many enzymes; Magnesium is a co-factor for many DNAmodifying

enzymes.

Na: Na was used as NaCl which provides ionic strength & helps in cell wall destruction.

It also helps to ppt. DNA by salting in process.

Isopropanol: It is used to precipitate DNA from dilute silutions which can be isolated by

centrifugation.

80% Ethanol: It is used to remove any excess salt by washing with it (pellet).

As shown in the photo of gel, it was broken while staining and destaining & during

handling. Still continuous DNA band was detected in the loaded sample under UV. This

proves the existence of DNA in isolated sample. RNA smear was detected at the bottom of

the gel as no RNase was used to remove RNA. Poor quality of agarose is the other

reason for the brakeage of the gel.

EDTA : Cells must be resuspended in buffer (some protocols call for washing the cells in the

buffer---i.e. resuspending and centrifuging---2 or 3 times) in order to have the ionic strength of the

solution compatible with biomolecules, in particular, the salt and pH. The EDTA is a chelating

agent that ties up divalent metal ions; these ions are often cofactors necessary for the action of

DNAses---enzymes present in the cell, which, when released by lysis along with the DNA, can

degrade the DNA.

SDS: SDS is an ionic detergent which will lyse most cells and denature some proteins.

Phenol-chroloform: Chloroform is used to deproteinize . The chloroform causes surface

denaturation of proteins; the isoamyl alcohol reduces foaming, aids separation and maintains the

stability of the layer of the centrifuged, deproteinized solution.

TE buffer : DNA must become rehydrated completely before it will form a uniform suspension.

This can take a long time, depending on the concentration of the DNA. It can be facilitated by

starting off with low ionic strength buffer and, once resuspended, adding an appropriate amount of

concentrated buffer stock to bring the buffer to the correct ionic strength.

Ethanol : Ethanol lowers the effective water concentration, causing large biomolecules to

interpenetrate and aggregate. The result is a visible precipitate at the interface, where the ethanol is

concentrated. As DNA is precipitated and removed, more is exposed to the ethanol and will

precipitate.