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Lab report 6 Biology SB015

Biologi (sb02), kolej matrikulasi perak.

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Experiment 6: isolating dna.

Objectives: At the end of this lesson, students should be able to isolate DNA from plant tissue.

Materials: Ice-chilled 95% alcohol Ice cubes Kiwi/ banana/onion/tomato/watermelon 0 g ethylenediaminetetraacetic acid (EDTA) 50 g sodium dodecyl sulphate or sodium lauryl sulphate (SDS or SLS) 8 g sodium chloride 4 g sodium citrate 1 litre water

Apparatus: 500 ml beaker Boiling tube Boiling tube rack Mortar and pestle Muslin cloth Water bath (60°C)

Procedures and Observation:

1. the salt-detergent solution is prepared. to completely dissolve the, salt without producing foam stir gently., 2 ml of ice-chilled alcohol is poured into a boiling tube and it is, placed into a beaker containing ice cubes., 3. kiwi/ onion/ tomato/ banana/ watermelon is peel, sliced and mash, with mortar., 4 fruit is transfered into a beaker and 100 ml of the salt-, detergent solution is added. the mixture is incubated in the water, bath at 60oc for 15 minutes., 5. the mixture is sieved after 15 minutes, with muslin cloth and the, liquid in a beaker is collected., 6. the boiling tube is filled half with sieved liquid., 7. 10ml of ice-chilled alcohol is poured very carefully into the side of, the boiling tube at a flat angle., 8 boiling tube is put into a rack and observed. observe and draw, the extracted dna between alcohol and the sieved liquid is observed, and drawn. crude dna has been found in between the alcohol and, sieved liquid., dna isolating is the crucial step in the identification of, dna procedure consists of lysis or the breakdown of cell, wall or membrane to release the dna using salt-detergent, in this experiment,the fuits that we use is banana,onion,and, tomato. this experiment we rupture fruits cells, thus, releasing their contents such as protein, dna, rna, lipids,, ribosomes and various small molecules. dna is then, suspended by alcohol as supernatant layer that we can see in, the boiling tube that contain different extract of plant, which, is banana,onion and tomato observation that we, got,dna of banana is more clear than tomato and onion, because banana is has soft texture that not need to mash, harder than tomato and onion. we assume that dna, structure of tomato and onion is has been damage while we, mash them in mortar., the precaution step that can be taken is make sure both, liquids do not mix and alcohol form a separate layer on top of, the sieved liquid should not mash the fruits harder to, avoid the dna is damaged..

Conclusion Plant tissue can be isolate by breakdown of cell wall or membrane to release the DNA using salt-detergent solution to remove proteins that are bound to the DNA. It also helps to keep the proteins dissolved in the aqueous layer so they don't precipitate in the alcohol along with the DNA.

1. What is the purpose of using the following?

A. salt-detergent solution, remove proteins that are bound to the dna keep the, proteins dissolved in the aqueous layer so they don't, precipitate in the alcohol along with the dna., b. ice chilled alcohol, helps the dna to precipitate more quickly., c. water bath at 60oc, to increase the rate of reactions to unzip the double helix, to separate the dna from the proteins to cause the dna to, precipitate out to cut the dna into fragments., 2 do we need to mash the fruits, exposes a greater surface area from which to extract the dna..

Multiple Choice

Course : biologi (SB02)

University : kolej matrikulasi perak.

precaution step in isolating dna experiment

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EXP 6: Isolating DNA

Isolating dna.

DNA is found inside the cells of all living things. In animals, plants and fungi most of the DNA is found inside the nucleus – the information centre of the cell.

This activity will enable you to extract DNA from fruit using basic household ingredients such as washing-up liquid, salt and vodka. You will be using essentially the same chemicals and processes that are used when DNA is extracted in the lab!

Students extract a visible mass of DNA from 4 different fruits and visually compare the amount of the DNA collected. Attached readings and activities will illustrate the structure and function of DNA. This activity is designed to give students basic concepts that are vital for understanding agricultural biotechnology: 1. Understand DNA is in the cells of all living organisms including in the foods we eat. 2. Determine how each of the ingredients in the protocol help extract DNA. 3. DNA can be extracted from cells for scientific use. 4. Extract a visible mass of DNA from fruit tissue

DEMONSTRATION VIDEO

LABORATORY BASED EXPERIMENT

HOME- BSED EXPERIMENT

HOME BASED EXPERIMENT PROCEDURE

REFERENCES

Experiments | Botany Practicals - Isolation of DNA from plant materials | 12th Botany : Practicals

Chapter: 12th botany : practicals, isolation of dna from plant materials, experiments, exercise : isolation of dna from plant materials.

DNA is one of the nucleic acids found in living systems. DNA acts as the genetic material in most of the organisms.

Principle: Recombinant DNA technology has allowed breeders to introduce foreign DNA in other organisms including bacteria, yeast, plants and animals. Such organisms are called Genetically Modified Organisms (GMOs). Thus rDNA technology involves isolation of DNA from a variety of sources and formation of new combination of DNA.

To isolate DNA from available plant materials such as spinach leaves, fresh green pea seeds, green papaya, etc.

Requirements:

Plant materials, mortar and pestle, beakers, test tubes, ethanol, etc.

Take a small amount of plant material and grind it in a mortar with a little amount of water and sodium chloride.Make it into a solution and filter it. To this filterate, add liquid soap solution or any detergent solution and mix it with a glassrod. Then tilt the test tube and add chilled ethanol and leave it aside in the stand. After half-an-hour we can observe the precipitated DNA as fine threads. DNA that separates can be removed by spooling DNA that separates can be removed by spooling

Observation:

DNA appears as white precipitate of very fine threads on the spool.

Thus DNA can be isolated from the plant cell nucleus by this technique.

Precautions:

· All the glasswares must be thoroughly cleaned and dried.

· The chemicals used for the experiments must be of standard quality.

· If ordinary ethanol is used, the time duration for obtaining precipitated DNA may extend further.

Isolation of Eukaryotic DNA: Objective, Principle, Requirements, Procedure, Precautions

Animesh sahoo.

September 27, 2021

DNA (Deoxyribonucleic acid) isolation is the process by which DNA is extracted from proteins, membranes, and other cellular materials contained in the cell.

In Eukaryotic cells (Human, plant, and animal cells), DNA is organized as chromosomes in the nucleus.

Table of Contents

Isolation of Eukaryotic DNA from tissues of a plant using the CTAB (cetyltrimethylammonium bromide) method.

Principle of Isolation of Eukaryotic DNA

DNA is a high molecular weight macromolecule, organized as chromosomes mainly in a eukaryotic cell organelle called a nucleus. Several methods are used for the isolation of eukaryotic DNA from tissues of organisms such as humans, plants, and animals.

Although, isolation of quality DNA from plants (mainly alkaloid-yielding plants) is rather difficult(due to the low yield of DNA high amount of plant tissues is required). The cesium chloride (CsCl) gradient method is expensive and time-consuming.

Therefore, the Cetyltrimethylammonium bromide (CTAB) method is commonly used due to the less expensive and high yield of eukaryotic DNA.

Requirements for Isolation of Eukaryotic DNA

Water bath incubator (60°C), refrigerated high-speed centrifuge
Plant leaves (as per desire and availability), mortar, and pestle.
Isopropanol and cetyltrimethylammonium bromide (CTAB)
Chloroform: isoamyl alcohol (24:1, v/v) (prepare fresh and keep in a dark bottle)
7.5 M ammonium acetate (57.8 g/100 me, pH- 7.7)
RNase (10 mg/ml with distilled water) or TE buffer (store in -20°C) [Tris-EDAT buffer (TE buffer)] [Tris-HCL 10mM (pH- 7.4) and EDTA 1mM (pH- 8.0)]
10mM ammonium acetate (0.0771 g/100ml, pH- 7.7 autoclave and store in refrigerator)
70% Ethyl alcohol (ethanol)

Preparation of isolation buffer

10 mM Tris-HCL (1 M stock solution, 12.11 g/100 ml, pH- 8.0)
(v/v) mercapteoethanal solution (should be added freshly)
M NaCl (29.22 g dissolve in 100ml to get 5 M stock solution)
mM EDTA (18.62 g/100 ml, pH-8.0, 0.5 M stock solution)
w/v CTAB (20 g/100 ml to get 20% stock solution)

Mix these chemicals fresh in a proper ratio and prepare an isolation buffer. For making a 10 ml isolation buffer mix these stock solutions in the following:

10 mM Tris-HCL- 0.1 ml
0.2% (v/v) mercapto-ethanol (fresh)- 0.02 ml
1.4 M NaCl- 2.8 ml
20 mM EDTA- 0.4 ml
2% w/v CTAB- 1 ml
Collect fresh plant tissue, weigh 1.0 g, and grind in liquid nitrogen to make powder or paste keeping in a pre-chilled pestle and mortar (liquid nitrogen may also be used). The plant tissues should not have been ground earlier.
Warm 10 ml isolation buffer in a centrifuge tube at 60°-65°C in a water bath.
Mix paste or powder of tissue in preheated isolation buffer. Incubate at 65°C in a water bath with gentle swirling.
Extract with equal volume (24:1) of chloroform and isoamyl alcohol and then mix gently.
Centrifuge at 10000 rpm for 20-25 minutes at room temperature.
Collect the clear aqueous phase (supernatant) very carefully using a wide bore pipette in a fresh centrifuge tube. Note the volume of the aqueous phase (the same process of chloroform and isoamyl alcohol extraction is repeated if the supernatant is not clear.
Gently add 2/3 volume of isopropanol to the aqueous phase and mix properly (the fibers of nucleic acid become visible).
Then centrifuge immediately for 10-15 minutes at 10000 rpm.
Remove the aqueous phase and collect over tissue paper so that isopropanol could be removed.
Wash the pellet twice with 70% ethanol centrifuging at 6000 rpm for 5 minutes at room temperature.
Dry the plates at 35°C for 30 minutes till completely dried.
Pour 100-200 ml TE buffer (for DNA isolation about 100 ml for 1 g tissue) or sterile distilled water so that the pellets could be dissolved. Add 10 ml RNase from the stock solution (10 mg/ml). Incubate for 30-60 minutes at 35°C.
Then add 2 ml of TE buffer or sterile distilled water to dilute the sample. Gently add 7.5 M ammonium acetate to get the final concentration of 2.5 M. Also adds 2.5 ml of cold ethanol for precipitating the DNA.
Incubate it overnight at -20°C (deep freeze) and centrifuge DNA at 10000 rpm for 15 minutes at 4°C.
Dry the pellets in the air and re-suspended them in 100-150 ml sterile distilled water or TE buffer.

A white or milky precipitate of DNA will be observed on the sides or bottom of centrifuge tubes after precipitating with iso-propanol and congratulation.

Precautions

EDTA, NaCl, CTAB, Tris-HCL, and ammonium acetate should be autoclaved.
All glassware should be autoclaved.
Chloroform: isoamyl alcohol (24:1) should be freshly prepared and stored in a dark bottle.

Animesh Sahoo is a scientific blogger who is passionate about biology, nature, and living organisms. He enjoys sharing his knowledge through his writings. During his free time, Animesh likes to try new activities, go on adventures, experiment with different biological aspects, and learn about various organisms.

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Eukaryotic DNA Isolation Protocol

Deoxyribonucleic acid (DNA) extraction is the process by which DNA is separated from proteins, membranes, and other cellular material contained in the cell from which it is recovered.
In eukaryotic cells, such as human and plant cells, DNA is organized as chromosomes in an organelle called the nucleus.
These cells also have a lipid bilayer outer membrane and cytoplasm containing proteins, sugars, lipids, and inorganic ions of various types and function. Eukaryotic cells also contain other membrane-enclosed compartments called organelles.
Hence, for the isolation of DNA from these cells, the three basic steps necessary include:

2) Precipitation

3) Purification

Table of Contents

Interesting Science Videos

Principle of Eukaryotic DNA Isolation

The first step in DNA isolation is the cell lysis in which the cell and the nucleus are broken open to release the DNA inside. This can be achieved be either mechanical disruption methods (using a tissue homogenizer (like a small blender), a mortar and pestle, by cutting the tissue into small pieces) or through lysis using detergents and enzymes such as Proteinase K to free the DNA and dissolve cellular proteins. An extraction buffer is often employed in the process consisting of 50mM Tris, pH 8, 25mM EDTA, 200 mM NaCl, 1% SDS etc.

The Tris buffer helps in pH maintenance.
EDTA binds divalent metal ions (Ca2+, Mg2+, Mn2+) that could form salt with anionic PO43-group of DNA. It also destabilizes the cell membrane, prevents precipitation of DNA and inhibits DNAses.
NaCl help loosen the cell wall for increased solubility and stability of DNA. It also enables DNA to precipitate out of an alcohol solution because it shields the negative phosphate end of DNA, causing the DNA strands to come closer together.
SDS which is an anionic detergent disrupts ionic interaction between proteins.
Liquid detergent causes cell membranes to break down by emulsifying the lipids and proteins of the cell and disrupting the bonds that hold the cell membrane together. The detergent also causes the lipids and proteins to precipitate out of the solution.
DNA released from disrupted cells is finally precipitated by cold absolute ethanol or isopropanol.
DNA is soluble in dish detergent solution but insoluble if ethanol is added. Adding alcohol precipitates the DNA that is it causes every component in the filtrate to stay in solution except the DNA.

Materials and Reagents of Eukaryotic DNA Isolation

5mL microcentrifuge tubes
Water bath 80 o C
Isopropanol (room temperature)
70% ethanol (room temperature)
Lysis solution
RNAse solution
Protein Precipitation Solution
Absorbent paper

Procedure of Eukaryotic DNA Isolation

Depending upon the sample at hand (animal cell or plant cell), perform cell lysis.
Add 3µL of RNase solution to the lysate
Invert the tube 2-5 times to mix sample
Incubate mixture at 37 o C (15-30 min)
Cool to room temp for about 5 minutes
Add 200µL of protein precipitation solution and vortex at a high speed (20 seconds).
Chill sample for about 5 minutes
Centrifuge at 13,000-16,000 x g (4 min)
On formation of a white pellet, remove the supernatant.
Transfer DNA in 1.5mL microcentrifuge tube w/ 600µLof room temp. Add isopropanol into the tube for precipitation.
Invert tube gently to mix solution.
White thread- like strands of DNA form a visible mass
Centrifuge at13,000-16,000 x g (at room temperature for 1 min)
On formation of a small white pellet, decant the supernatant
Add 100µL of room temp.70% ethanol
Invert tube several times (washing of the DNA)
Centrifuge at13,000-16,000 x g ( at room temperature for1 min)
Aspirate ethanol
Invert tube on a clean absorbent paper
Air-dry pellet (10-15 minutes)
Add 100µL of DNA Rehydration Solution
Incubate at 65 o C for 1 hour (Rehydration of DNA)
Store DNA(2-8 o C)
DNA concentration can be determined measuring the intensity of absorbance of the solution at the600 nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations. DNA absorbs UV light at 260 and 280 nm while proteins absorb UV light at 280 nm.

Expected Results of Eukaryotic DNA Isolation

White thread like strands or white pellet of DNA

On Spectrophotometry,

Pure DNA = 1.8
DNA with protein < 1.8
https://www.ncbi.nlm.nih.gov/pubmed/3037944
https://www.sciencedirect.com/topics/neuroscience/dna-extraction
https://bioprep.community.uaf.edu/learning-modules/2-dna-extraction-4/the-basics-of-dna-extraction/
https://www.scribd.com/doc/21423188/Isolation-of-DNA
https://www.encyclopedia.com/science-and-technology/biology-and-genetics/genetics-and-genetic-engineering/dna-isolation-methods
https://www.scribd.com/document/44958187/DNA-Extraction
http://virtuallibrary.stao.ca/sci-tie-data/lessons/biology/b12/DNA_EXTRACTION_EXPERIMENTans.html

About Author

Sagar Aryal

NOTIFICATIONS

Dna extraction.

+ Create new collection

DNA extraction is a routine procedure used to isolate DNA from the nucleus of cells.

Scientists can buy ready-to-use DNA extraction kits. These kits help extract DNA from particular cell types or sample types. However, they can be expensive to use routinely, so many labs have their own methods for DNA extraction.

Double-stranded DNA.

The long stringy nature of DNA is hard to conceptualise. By extracting it, the concept can become easier to understand. This activity outlines how to extract the DNA from a tomato.

What does DNA extraction involve?

Step 1. breaking cells open to release the dna.

The cells in a sample are separated from each other, often by a physical means such as grinding or vortexing , and put into a solution containing salt. The positively charged sodium ions in the salt help protect the negatively charged phosphate groups that run along the backbone of the DNA.

A detergent is then added. The detergent breaks down the lipids in the cell membrane and nuclei . DNA is released as these membranes are disrupted.

Step 2. Separating DNA from proteins and other cellular debris

To get a clean sample of DNA, it’s necessary to remove as much of the cellular debris as possible. This can be done by a variety of methods. Often a protease ( protein enzyme) is added to degrade DNA-associated proteins and other cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the sample.

Step 3. Precipitating the DNA with an alcohol

Finally, ice-cold alcohol (either ethanol or isopropanol ) is carefully added to the DNA sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. By gently stirring the alcohol layer with a sterile pipette, a precipitate becomes visible and can be spooled out. If there is lots of DNA, you may see a stringy, white precipitate.

DNA precipitate

When an ice-cold alcohol is added to a solution of DNA, the DNA precipitates out of the solution and if there is enough DNA in the solution, you may see a stringy white mass.

Step 4. Cleaning the DNA

The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer and ready to use.

Step 5. Confirming the presence and quality of the DNA

For further lab work, it is important to know the concentration and quality of the DNA.

Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of DNA in a sample. Alternatively, gel electrophoresis can be used to show the presence of DNA in your sample and give an indication of its quality.

What can this DNA be used for?

Once extracted, DNA can be used for molecular analyses including PCR, electrophoresis, sequencing, fingerprinting and cloning.

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COMMENTS

Lab report 6 Biology SB015 - EXPERIMENT 6: ISOLATING DNA ...
and drawn. Crude DNA has been found in between the alcohol and sieved liquid. Dicussion: DNA isolating is the crucial step in the identification of DNA procedure consists of lysis or the breakdown of cell wall or membrane to release the DNA using salt-detergent solution. In this experiment,the fuits that we use is banana,onion,and tomato.
Precautions during Nucleic Acid Extraction for NGS Work
Precautions to be taken during DNA isolation: 1. Make sure that the vials and tips are DNase free. 2. Avoid vortexing during DNA extraction. 3. Make sure that the extracted DNA is treated with RNase to remove the RNA. 4. It is good to use the columns to purify the DNA. The columns will remove the salts presents in the DNA. 5.
Experiment: DNA Extraction from Fruit - FutureLearn
Experiment to purify DNA from fruit. Step 1: Mash up the fruit of your choice in a bowl. Bananas, kiwis and strawberries all work well. (Remove the skin of the bananas and kiwi, we just want the insides!) Step 2: In a separate bowl, mix the washing up liquid, salt and tap water. Stir gently trying to avoid making too many bubbles in the mixture.
AMALI BIOLOGI KMS - EXP 6: Isolating DNA - Google Sites
Experiment: DNA Extraction from Fruit An experiment highlighting how DNA can isolated from cells, using fruit to illustrate this principle. Extracting DNA from Fruit All living things have DNA. This hands-on activity will enable you to extract DNA from fruit, such as strawberries, using everyday household items.
Isolation of DNA from plant materials - Experiments | Botany ...
DNA appears as white precipitate of very fine threads on the spool. Inference: Thus DNA can be isolated from the plant cell nucleus by this technique. Precautions: · All the glasswares must be thoroughly cleaned and dried. · The chemicals used for the experiments must be of standard quality.
Isolation of Eukaryotic DNA: Principle, Requirements, Procedure
Sep 27, 2021 · Principle of Isolation of Eukaryotic DNA. DNA is a high molecular weight macromolecule, organized as chromosomes mainly in a eukaryotic cell organelle called a nucleus. Several methods are used for the isolation of eukaryotic DNA from tissues of organisms such as humans, plants, and animals.
Expt 1. Isolation of DNA from banana Aim: Materials Required
Spool the DNA with a glass rod or needle. Observation: The DNA appears as white precipitate of fine thread on the spool. Precaution: All the glassware used must be thoroughly cleaned and dried. The enzyme and other chemicals used must be of standard quality.
Eukaryotic DNA Isolation Protocol - Microbe Notes
Aug 3, 2023 · The first step in DNA isolation is the cell lysis in which the cell and the nucleus are broken open to release the DNA inside. This can be achieved be either mechanical disruption methods (using a tissue homogenizer (like a small blender), a mortar and pestle, by cutting the tissue into small pieces) or through lysis using detergents and ...
DNA extraction - Science Learning Hub
Step 4. Cleaning the DNA. The DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer and ready to use. Step 5. Confirming the presence and quality of the DNA. For further lab work, it is important to know the concentration and quality of the DNA.
Experiment #6: DNA Extraction from Fruits - KU
DNA is an incredibly small molecule, but in large quantities, it can be seen. In this activity, you will extract DNA from banana, strawberry and kiwi fruits. We are going to use fruits because they are soft and easy to pulverize. There are three basic steps in DNA extraction. First, the cell must be broken open to release the nucleus.