UNIVERSITI TEKNOLOGI MARAFAKULTI KEJURUTERAAN KIMIA

GENETICS LABORATORY (CBE 561)

NAME : NURWANI BINTI HUSSINGROUP : 4AEXPERIMENT : LAB 1 : GENES IN A BOTTLE DATE : 20 MARCH 2014PROG/CODE : EH222SUBMIT TO : MOHAMAD SUFIAN BIN SO’AIB

No Title Allocated Marks (%) Marks1 Abstract 52 Introduction 53 Objectives 54 Theory 55 Procedures/Methodology 106 Apparatus 57 Results 108 Calculation 109 Discussion 20

10 Conclusion 1011 Recommendations 512 References 513 Appendices 5

TOTAL 100

Remarks:

Checked by: Rechecked by:

Date: Date:

I. ABSTRACT

This experiment has provide the great tips on extraction of nucleic acid which is both DNA and RNA. These two nucleic acid store in the nucleolus of nucleus in a cell. Certain substances needed in order to make the nucleic acid settle out. Focus on the onions’ cell and how it works. The substance like salt, detergent and alcohol added performed different function.

The smashed and chopped onion used has significantly gave different the result. Both onion used to identify the correct way to get the extraction and understand the concept of DNA extraction.

II. INTRODUCTION

Human is made up of cell which perform as building block. In a single cell, there is an organelle called nucleus which play its role as a headquarters and control all activities inside that single cell. The activities are including amino acid synthesis and break down of the glucose molecule etc. Inside nucleus, there is nucleolus where it store the genetic information of DNA. These genetic information have a special function which are responsible for expressing someone physical characteristic such as eye’s color, height and skin color. DNA also carries information that will be required for the cells’ to function which mean the cells act based on the genetic information carried by DNA.

The DNA has a structure of double helix where it is made from two strands and spiral shape. It consist of bases such as A, T, G and C which represent as adenine, thymine, guanine and cytosine. Adenine pairing with thymine and guanine pairing with cytosine that hold by hydrogen bonds and form base pairs. The number of base pairs is numerous in a DNA. Sugar phosphate backbone hold the base tightly that actually has produced a complete DNA.

Figure 1: Illustration of DNA

The letters A, T, G and C that actually form base pairs to code up genetic information in a DNA. But the arrangement is different for every species. Even for every human, the different in arrangement make the criminal scene easy for the detectives to understand or imagine what was actually happen and detect the criminal with flying colors. This is because even a letter in the arrangement is different with another person. That is why everybody has unlikely to have same skin color or height. The base pairs arranged to give a message that contain an information where the cells called genes can read it and perform a certain function. The DNA located in the nucleus found to be folded and molded by protein in a cell.

Isolation genetic materials done by scientist in order to understand certain characteristic of a species. The study of DNA is usually for detecting bacteria and viruses in surrounding environment and

also the diagnosing disease and genetic disorders. The techniques of DNA extraction are also various in ways.

III. OBJECTIVES i. The objective of this experiment is to isolate genetic materials from onion.

ii. To understand the concept of protein digestion.iii. To precipitate DNA with cold alcohol.

IV. THEORY

Isolation of genetic materials or DNA from onion can be done in various way. Certain needed substance added during experiment for a certain reason. For example, the addition of salt helps the DNA to precipitate when the alcohol is added. That is why people who drink alcohol are not recommended to eat salty food which can cause high blood pressure. The fact is the backbone of the DNA which is deoxyribose sugar linked by phosphodiester group. In the neutral pH water, oxygen atom bonded to phosphorus negatively charged. The negativity makes the DNA dissolved in water because high polarity. Therefore, after addition of enough salt, the positively charge cation is equal in number with anion bonded to phosphodiester group that make the DNA less polar to water which actually slowly becomes less soluble to water and tend to precipitate out. In a simple sentence, the negative charge neutralize by positive charge of salt and DNA become less polar and cannot dissolve in water.

Theoretically by comparing smashed and chopped onion, the smashed onion is more significantly really work for the experiment. Onion surrounded by cell wall can be broken by smashing process unlikely chopped onion. When the cell wall already broken, now it is easy for the detergent to play its role to lyse the DNA in the nucleus. The DNA located in nucleus folded and molded by protein and lipid of nucleus membrane. In order to expose the DNA into water, the membrane must be broken down by using detergent. Detergent that contained the compound SDS (sodiumdodecyl sulfate) play its role to break the lipid molecules and protein membrane where DNA finally lyse into water. Chemically, detergent contain enzymes that would denature the structure of protein and release all nucleic acid because later on the detergent will form bond to the protein or lipid. Nucleic acid can be classified as DNA and RNA. The amount of detergent use in the experiment must be in correct amount, overdose amount of detergent make it hard to control the bubble formation and the worst part is the cell would destroyed.

Figure 2: The detergent captured the protein and lipid of the cell membrane

Another substance that make up this experiment is alcohol. Alcohol make the DNA to precipitate or settle out of the solution. The DNA will leave all the cellular components that are not soluble in alcohol. DNA formed can be out from the solution using stirring rod. The cold water is better than warm water for extraction because cold water helps the DNA intact during the extraction process. Cold water will slow down the cellular activity and preventing the enzyme from being destroy. Most of enzyme will undergo denaturation at the temperature of above 40 DC and the optimum temperature for enzyme is around 37 DC.

Figure 3: The activity of enzymes against temperature

V. PROCEDURES/METHODOLOGY 1) 100ml of mix solution containing 10ml detergent (Dynamo), 90ml distuilled water and

1,5g of table salt was prepare in a 250ml beaker.2) The onion was cut and smashed with pestle and mortar.3) The smashed onion mixed with 100ml mixture solution.4) Water bath was prepared at the temperature of 55-60 ℃.5) The solution beaker was placed in the water bath for 10-12minutes.6) After 10-12minutes in water bath, the beaker then was placed in the water bath for

5minutes.7) The solution was filtered into two ways, first way solution was filtered by filter paper

which shaped into a cone and pass through into a conical flask and the second way of filtration was manually using hand which also pass through a conical flask.

8) Isopropyl alcohol was measured as 2.5ml and was poured into a vial container and labelled as 1 and 2.

9) The labelled 1 and 2 represented the size of onion particles, which vial 1 contained the onion solution that was smashed by pestle and mortar and vial 2 contained onion solution that was being cut into cube small pieces.

10) Vial 1 and 2 was placed in a centrifugal machine for 5minutes for completing the separation step.

11) Any changes that was being observed have been identified and recorded in order to discuss it later.

VI. APPARATUS

Apparatus Function Image 1) Pestle and mortar To smash the onion and

make it into smaller particles

2) Vial bottle To store or keep the sample and use it later in the centrifugal machine

3) Detergent Break down the cell membrane by dissolving the lipids and proteins of the cell by disrupting the bonds that hold the cell

membrane together

4) Onion As the subject of this

experiment where the DNA of this onion being

extracted

5) Centrifugal machine

Use to separate materials suspended in a liquid medium based on

the response to the gravity.

6) Conical flask and funnel

Multifunction in laboratory but more easy when there is

filtration happen where the filter paper easily place on the funnel so

that the solution filtrated well.

7) Filter paper Used to remove solid particles in the mixture of onion. It comes from

various sizes and can purchased what size

particles the paper can remove.

VII. RESULT

Smashed by pestle and mortar Chopped onionImage

Observation Seen a black thread-like DNA DNA not detectedExplaination The black thread-like recorded

was confirmed as DNA by the lecturer. Extraction DNA of

onion successfully recorded as the particles of the onion

already break into really small and easy for DNA to isolate

from solution.

The DNA cannot be seen in this case because the size of onion was not small enough.

VIII. CALCULATION

No calculation.

IX. DISCUSSION

Based on the result tabulated, this experiment is successfully come out with the DNA. The black thread-like was the DNA observed at the bottom vial. Onion DNA supposed to have a color of white but perhaps during collecting the sample, the apparatus in the laboratory is not clean enough. The solution contained onion initially added by salt which promotes the neutralization of negatively charge ion into positive ion which cause the DNA start to precipitate out.

Strength of detergent is various but in this experiment Dynamo brand was used in order to promote enzyme in the detergent to break the protein and lipid of the nuclear membrane. Enzyme in detergent called SDS works as the meat tenderizer. They have the same role in this experiment. The exact strength scale of the detergent is not available but it is estimated suitable for this experiment. Unlike the Am Way brand, only a small amount like 1ml is enough. Because of insufficient of exact strength scale of detergent, perhaps the DNA come out with black color because of the chemical inside the detergent.

Temperature of water bath likely not beyond the boiling point because the enzyme from the detergent only can tolerate until the temperature of 40 DC or the enzyme will eventually denatured. Filter paper also play an important role, because if the filter paper too thick, it would trap the DNA and never drained into the conical flask. If there is no filter paper, manually filter using hands is preferred.

The alcohol added to the onion solution which promote the DNA to precipitate out. Table salt and alcohol is simply to have the same function but differ in mechanism and works together to make this experiment really success. The solution once soaked in cold water in order to reduce cellular activities and help DNA intact during extraction process.

The successful experiment is done by smashed onion, contrast with chopped onion. Chopped onion does not show any DNA available because it is so hard for the salt or detergent to break the cell wall. Compare to smashed onion, it already been given a strong force using pestle and mortar to break the cell wall of the onion which then salt and detergent can carry their role.

X. CONCLUSION

In a nut shell, it can be concluded that, the onion need to be smashed up in order to get the precipitate of DNA sequence. Compare to chopped onion, the result will be disappointing where the cell wall cannot be penetrated by detergent and salt. Insufficient data resulting the DNA to have a different color which are not the true color of onion’s DNA.

This experiment is actually the extraction of nucleic acid which is both DNA and RNA. Initially these two type of nucleic acid has a long and thin physical characteristic in a nucleus but after the addition of alcohol, they clump together and precipitate out using salt right after the nucleus membrane broken by the enzyme found in the detergent. Cold water, slow down the activities in the cell and make everything easy to work on.

XI. RECOMMENDATION

1. During soaking the beaker in the hot water bath, the temperature must be not too hot or overtime as referred to the procedure because the hot temperature will destroy the enzyme.

2. The amount of detergent must be measured depends on strengthen of the detergents itself.

3. The onion must be smashed nicely, if the onion is over smash, the cell will destroyed because the force and smash process breaks the cell wall.

4. Filter paper used in the experiment must be suitable with the particles size of the onion because thick filter paper will trap the cellular content and also the DNA needed for the experiment.

5. Make sure all the apparatus used in the experiment is clean and trusted enough to proceed the experiment because dirty apparatus will produce inaccurate result.

XII. REFERRENCE

1. Centrifugation Basics. (2002). Retrieved from Department of Biology of Bates College: http://abacus.bates.edu/~ganderso/biology/resources/centrifugation.html

2. (1994). DNA EXTRACTION. The Gene School.

3. Holum, J. (1968). Elements of General and Biological Chemistry.

4. How to Extract DNA from Anything Living. (2012, January 24). Retrieved from Genetic Science Learning Center: http://learn.genetics.utah.edu/content/labs/extraction/howto/

5. Kim, J. (2013, Feb Tuesday). 10 Questions You Want To Ask About Proteinase K. Retrieved from AG SCIENTIFIC: http://info.agscientific.com/blog/bid/158711/10-Questions-you-want-to-ask-about-PROTEINASE-K

6. Pierce Protein Biology Products. (n.d.). Retrieved from Thermo Scientific.

7. Skipor, A. (2012, June). NEWTON. Retrieved from Filter Paper: http://www.newton.dep.anl.gov/teachers/resources/basiclab/filter.html

XIII. APPENDICES