FACULTY OF RESOURCE SCIENCE AND TECHNOLOGY

STB2163 - RECOMBINANT DNA TECHNOLOGY

LABORATORY REPORT 1 :

GROUP NUMBER 9

GROUP MEMBERS LIST

NUM. NAME MATRIC

NUMBER

1. MALISSA BINTI KAMARUDIN (56546)

2. S.UVANAPPRIA A/P SATHASIVAM (58017)

3. MOHD AMIRUL FIRDHAUS BIN MOHD RIDHWAN (58740)

4. MUHAMMAD QAYYUM BIN ZAMAN HURI (58842)

DECLARATION
We declare that this material, which we now submit for assessment, is entirely our own work
and has not been taken from the work of others, save and to the extent that such work has been
cited and acknowledged within the text of our work. We understand that plagiarism, collusion,
and copying are grave and serious offences in the university and accept the penalties that would
be imposed if we engage in plagiarism, collusion or copying.
 Lecturer Lecturer at Faculty of Resource Science and Technology
University Malaysia Sarawak.

Due Date 22 March 2018

For Lecturer’s Use

Received Date

Mark

Introduction

Practical 1 : Introduction to the molecular biology laboratory

Analysis of the cell component such as DNA involves the use of specialty equipment,

techniques, and protocols. Standards equipment used in the molecular biology laboratory

includes the use of micropipettes,microcentrifuge,microwave oven , water bath, pH meter as well

as other basic tools.

Practical 2 : Isolation of Chromosomal DNA from Bacteria Culture

Many different methods and technologies are available for the isolation of genomic or

chromosomal DNA. In general , all methods involve disruption and lysis of the starting material

followed by the removal of proteins and other contaminants and finally recovery of the DNA.

Removal of proteins is typically achieved by digestion with proteinase K, followed by salting-out

, organic extractioin, or binding of the DNA to a solid-phase support. DNA is usually recovered

by precipitation using ethanol or isopropanol. The choice of a method depends on many factors:

the required quantity and molecular weight of the DNA, purity required for downstream

applications , the time and expense.

Genomic or chromosomal DNA extraction is the simpler procedure becausee all that is

needed is a good strong lysis to release the genomic DNA into solution. For yeast, plant cells and

bacteria , this involves breaking down the strong, rigid cell wall before mechanically disrupting

the membrane. The cell wall can normally be broken down using enzyme such as lysozyme

which catalyses the hydrolysis of the cell wall peptidoglycans and the serine protease, proteinase

K. Cell wall debris , polysaccharides , and remaining proteins are removed by selective

precipitation with CTAB, and high-molecular-weight DNA is recovered from the resulting

supernantant by isopropanol precipitation. Once the sample has been lysed so bringing the

genomic DNA into solution, all that is needed is to purify the sample. This can be achieved using

either phenol-chloroform or a spin filter membranes by adding guanidine salts that promote

binding to silica.
Objectives

Practical 1 : Introduction to the molecular biology laboratory

1. To get familiarize with common techniques used in the molecular biology laboratory
2. To get the practice of proper pipetting technique, centrifuging, and following an

experimental protocol

Practical 2 : Isolation of Chromosomal DNA from Bacteria Culture

 1. To understand the biochemical and molecular effects of each reagent used in the isolation

of chromosomal DNA protocol

2. To understand the difference in mobility between chromosomal DNA fragments when

analyzed on agarose gel.

EXPERIMENT 1A: PROPER USE OF MICROPIPETTES,MICROCENTRIFUGE AND

OTHER BASICS MOLECULAR WORK EQUIPMENTS.

Materials:

● A set of three pipettes per group (2-20 μL, 50-200 μL & 200-1000 μL)
● Three boxes of corresponding pipette tips per group (white,yellow & blue)
● Microcentrifuge tubes per group
● Beaker per group for pipette tip waste
● Soil suspension (for centrifuge & pipette practice)
● Latex gloves
● Microcentrifuge
● 6x gel loading dye
● Ficoll (type 400)
● 1% bromophenol blue (BPB)
● 100% glycerol
● Tris base
● Acetic acid glacial
● 0.5M Ethylenediaminetetraacetic acid (EDTA) solution
● Boric acid
● 0.25% bromophenol blue
● 0.25% xylene cyanol FF
 ● 15% Ficoll Type 400
● 120mM EDTA

Procedures:

a) Pipetting Technique
1. The most appropriate volume pipette and corresponding tip are chosen.
2. The volume on the pipette is adjusted by turning the knob to the desired volume.
3. The pipette is depressed. Pipette tip is inserted into liquid and released.
4. The pipette is depressed again to discharge the liquid being careful not to contaminate the

tip during transfer.

5. The pipette tip is released into desired tube by pressing white button.
6. Liquid solution of 1000 μL, 500 μL and 10 μL are pipetted. Use the right micropipettes is

ensured to adjust each type of micropipette to the right aspiration volume and the right

kind of micropipette tips is used.

7. 15 μL of solution is loaded into an agarose gel provided. The right proportion of 6x GLD

to solution is added. Solution for loading must contain 1x GLD.

b) Centrifuge Technique

1. Centrifuge tubes are placed directly across from each other in the centrifuge.
2. The tubes have similar volumes of sample and an even number of tubes are ensured.

c) Protocol for separating clear fluid from a cloudy mixture ( Pipette & Centrifuge Practice)

1. The soil solution mixtures are swirled and 500 μL from each mixture is aliquot into

labelled centrifuge tubes.

2. The mixture at 12,000 rpms is centrifuged for 1 minute.
3. Supernatant is pipetted carefully into a fresh labelled tube.
4. Steps 2 to 3 are repeated with the supernatant obtained until a clear liquid solution. All

tubes are repeated.

EXPERIMENT 1B: ISOLATION OF CHROMOSOMAL DNA FROM BACTERIAL

CULTURE

Materials:
 ● Overnight bacterial culture
● Tris-EDTA (TE) Buffer:

10mM Tris-CI pH 7.41 mM

Ethylenediaminetetraacetic acid (EDTA)

1 litre ddH20

● 10% sodium dodecyl sulfate (SDS)

● 20mg/ml proteinase K (stored in small single-use aliquots at -20°C)
● 5 M NaCI
● CTAB/NaCI solution (10% CTAB in 0.7M NaCI)
● Choloform/isoamyl alcohol (24:1)
● Phenol/choloform/isoamyl alcohol (25:24:1)
● Isopropanol
● 70% ethanol

Procedures:

1. A 5 mL liquid culture with the bacterial strain of interest is inoculated. The strain is grew

in appropriate conditions (medium,drug selection,temperature) until the culture is

saturated.
2. 1.5 mL of the culture is spinned in a microcentrifuge for 2 minutes. The supernatant is

discarded.
3. Pellet in 567 μL TE buffer is resuspended by repeated pipetting. 30 μL of 10% SDS and 3

μL of 20 mg mL-1 proteinase K is added to give a final concentration of 100 μg mL-1

proteinase K in 0.5% SDS. The solution is mixed thoroughly and incubated 10 minutes at
 37 °C.
4. 100 μL of 5 M NaCI is added and mixed thoroughly.
5. 80 μL of CTAB/NaCI solution is added. The solution is mixed thoroughly and incubated

10 minutes at 65 °C.
6. An approximately equal volume (0.7 to 0.8 mL) of choloform/isoamyl alcohol is

added,mixed thoroughly and spinned 4 minutes in a centrifuge.

7. Aqueous,viscous supernatant is removed to a fresh microcentrifuge tube,leaving the

interface behind. An equal volume of phenol/chloroform/isoamyl alcohol is

added,extracted thoroughly and spinned in a microcentrifuge for 5 minutes.

8. The supernatant is transferred to a fresh tube. 0.6 vol isopropanol is added to precipitate

the nucleic acids. The tube is shaked back and forth until a stringy white DNA precipitate

becomes clearly visible. At this point,the precipitate can be pelleted by spinning briefly at

room temperature.
9. The DNA is washed with 70% ethanol to remove residual CTAB and respinned 5 minutes

at room temperature to repellet it. The supernatant is carefully removed and briefly air

dried the pellet at room temperature. The pellet is redissolved in 100 μL TE buffer.
EXPERIMENT 1C: AGAROSE GEL ELECTROPHORESIS (AGE) ANALYSIS

Materials:

● TAE Buffer (50x)

● TBE Buffer (10x)
● Loading Dye Buffer (6x)
● Agarose powder
● Standard DNA Ladder marker
● Sterile ddH20
● 10 mg/ml ethidium bromide (EtBr)
● Agarose Gel Electrophoresis System
● DNA samples (bacterial plasmid & chromosomal DNA)

Procedures:

a) Preparation of Mini agarose-gels

1. The appropriate size of mini gel rig/comb combination based upon the number of

samples to be run is chosen.

 2. The gel mold is turned so that the gaskets are against the inner walls of the electrode

chamber. The comb in the mold is positioned.

3. The flask is heated with agarose gel solution using microwave.
4. The appropriate volume of hot agarose is measured into a graduated cylinder.

b) Preparation of samples and use of mini gels

1. The comb is gently removed by pulling evenly upward.

2. The gel mold is position in the electrode chamber and covered with gel running buffer.
3. Since the agarose gel is submerged beneath electrophoresis buffer, the samples are loaded

into the lanes below the meniscus of the buffer solution.

4. Using a permanent marker,the number of samples to be analysed is written on the

Parafilm. 10 μL of dye near each of the number is spotted. An extra spot is added for the

molecular weight standard (right) that should be used next to samples.

5. 4 μL of sample is pipetted and added to the dye spot of the correct number. The tip is held

and ejected. The tip is placed on another pipette set to 14 μL and drew up the entire

sample/dye mixture. The mixture is pipetted to the appropriate mini-gel lane so that when

photographed lane number one is to the top left. All the samples are continued and the

molecular weight standard DNA marker (MWM) is loaded.

6. The cover is placed to the electrode box and the wires are plugged in to the power pack.

The samples are migrated towards the red or anode pole. The power pack is turned on and

the low milli-ampherage setting is used to start the electrophoresis at 75 mA.

c) Staining and Photographing Mini-gels

1. After switching off the power,the gel mold is removed gently from the electrode chamber

and the gel is slipped into the plastic staining box containing EtBr solution.
2. The EtBr stock solution is usually kept at 4°C in a dark bottle as a 10 mg/ml solution. To

use, 20 μL of EtBr is added to 40 ml of 1x TBE buffer and stained for 15 minutes in the
 dark.
3. To view EtBr-stained DNA, the UV light is used. Protective eye goggles is wore all the

time. The room lights are switched off to view the gel.
4. To photograph mini-gel, the transilluminator is turned to the highest setting and

documented the agarose gel photo using digital camera. The agarose gel is disposed in the

biohazard waste bin. The surface of the transilluminator is wiped clean with distilled

water.

Results

Band 9
 Wells

Ladder

Figure 1

Discussion
 Gel electrophoresis is a technique that can separate and analyze any molecule. Based on

our experiment using E. Coli DNA, band in row 9 shows positive result with the presence of

DNA smear. Preparation of chromosomal DNA consist of lysis which is followed by incubation

with a nonspecific protease which is proteinase K and a series of extractions prior to

precipitation of the nucleic acids. These procedures effectively remove cell wall and denatured

protein mostly by the proteinase K treatment but not the polysaccharides complexes which can

interfere with enzyme activity. The incubation of proteinase K followed by Cetyltrimethyl

ammonium bromide (CTAB) solution effectively removes the subsequent and produce digestible

chromosome DNA. Remaining protein and CTAB solution was removed by phenol and

chloroform extraction. DNA can also be recovered and concentrated from the aqueous phase by

ethanol precipitation or through the use of a centrifugal filter, which allows for additional

purification and concentration of the DNA in the samples (Koons et al., 1994). These extraction

methods with a ratio of 25: 24: 1 respectively for phenol, chloroform and isoamyl alcohol.

Isomayl alcohol treatment helps to prevent the formation of foam in the sample. The DNA

fragment then filled with dyes in the agarose gel to easily visualize and facilitate the observation

of DNA fragment when exposed to UV light. In this experiment, we used ethidium bromide

reagent (EtBr) as the dyer. Therefore, we got a positive result for our group in row 9.

DNA sample were poured in the well combs of agarose gel and an electric current with

specific voltage was applied to move them through the gel. DNA fragments are negatively charge

because of their phosphate groups in their sugar-phosphate backbone which move the fragment

towards the positive electrode. The movement of protein through this gel depends on the charge

density (charge per unit of mass) of the molecules (Karp G, 2008). The small molecule moves
faster than larger ones. As our result, only a little amount of DNA smear that we can obtain

compared to the ladder. Smearing of DNA might be caused by overloading of sample in the well

of electrophoresis gel. The sample is not properly diluted and the excess sample may smear

across the gel. Overloading of sample also can caused the sample to spill out of the well if the

sample were moved from it first place. Smearing of DNA also can be caused by improperly

prepared sample. According to Hoff-Olsen,1999 , there are four commonly used extraction

procedures for DNA extraction. In this experiment we used organic extraction method including

SDS and proteinase K treatment. The procedure might not be done correctly where the enzyme

may break down the sample too much which resulting it to smear. Choosing the incorrect buffer,

temperature or pH also can cause the enzyme work improperly which produce smear band on the

electrophoresis gel.

To improve this experiment with a better result, precautionary step must be taken

especially during the pipetting procedure. For example, when transferring liquid samples, contact

with the tip of the pipette should be avoided. This is to avoid contamination that will affect the

results. It is important to pipette a correct volume of substances to prevent the excess interaction

between the solution and the sample. A disadvantage of this experiment is the use of phenol and

chloroform that are health hazard and harmful. Precaution step must be handle carefully and

properly dispose. Strataclean resin which is non-toxic and non-combustible silica particle is

recommended to be used instead of phenol and chloroform. It is used to clean up DNA or

concentrate the dilute protein. Another method which is using xanthogenate also helps to

increase the efficiencies of isolation of pure DNA from a small amount of bacterial cell. The

modification of the procedure describe by Jhingan (Jhingan,1992). Successive enzymatic lysis

such as proteinase K that we use in this experiment is time-consuming. Moreover this enzyme

lack efficiency on most gram positive bacteria while it is very efficient to the gram negative

bacteria (Grimberg. J and Sloan. G. L., 1986).

Conclusion
Practical 1 : Introduction to the molecular biology laboratory

In conclusion, students get familiarize with common techniques used in the molecular biology

laboratory . In addition , students get the practice of proper pipetting technique, centrifuging,

and following an experimental protocol

Practical 2 : Isolation of Chromosomal DNA from Bacteria Culture

Lastly , students able to understand the biochemical and molecular effects of each reagent used

in the isolation of chromosomal DNA protocol. Morever , students could understand the

difference in mobility between chromosomal DNA fragments when analyzed on agarose gel.

Reference

Karp G. Cell and Molecular Biology: Concepts and Equipments.5th edition. London, UK: John Wiley &
Sons; 2008.