LAB 6: EXTRACTION OF PLASMID DNA USING GF-1 PLASMID DNA EXTRACTION KIT

INTRODUCTION

 The  GF-1 Plasmid DNA Extraction Kit is designed for rapid and efficient purification of

high copy and low copy plasmid DNA without the need for precipitation or organic

extractions. This it uses a specially-treated glass filter membrane fixed into a column to

efficiently bind DNA in the presence of high salt. Combining alkaline lysis-SDS and minicolumn spin technology, up o 20μg of plasmid DNA from bacterial cultures can be isolated. Multiple samples can be processed rapidly and with practice, the purification takes less than 30 minutes. Optimized buffers ensure only highly pure plasmid DNA is extracted and is ready to use in many routine molecular biology applications such as restriction enzyme digestion, radioactive/fluorescence DNA sequencing, PCR, ligation, transformation and other

manipulations.

OBJECTIVE:
to extract plasmid DNA from plasmid-containing culture.

DISCUSSION :

Before the extraction started,dilute concentrate Wash buffer and washing steps are necessary to remove unbound reagents and reduce background, thereby increasing the signal:noise ratio. Insufficient washing will allow high background, while excessive washing may result in decreased sensitivity caused by elution of the antibody and/or antigen from the blot. Occasionally, wash buffer formulations consist of only a physiological buffer such as Tris buffered saline (TBS) or phosphate buffered saline (PBS) without any additives. More commonly, a detergent such as 0.05% Tween 20 is added to the buffer to help remove nonspecifically bound material. Depending on the specifics of the assay, the amount of detergent in the wash buffer will vary, though typical concentrations are from 0.05 to 0.5% for detergents like Tween-20*. Another common technique is to add a 1:10 dilution of the blocking solution to the wash buffer. Including the blocking agent with the detergent may help to minimize background in the assay by preventing elution of the blocking protein from the membrane and/or allowing nonspecific interactions to occur with the protein in solution rather than those immobilized on the membrane.

Ratio between the readings (OD260/OD280) provides an estimate of the purify of the sample. Pure plasmid DNA has ratio 1.8691. If there is contamination proteins,the OD260/OD280 will be significantly less. Meanwhile the ratio between the readings at (OD260/OD230) evaluates the level of salt carryover in the plasmid DNA.The OD260/OD230 is best if greater than 1.5 and the result is 1.6376.Lower the ratio if greater the amount of salt that is present. 

 In a spectrophotometer, a sample is exposed to ultraviolet light at 260 nm, and a photo-detector measures the light that passes through the sample. Using the Beer Lambert Law it is possible to relate the amount of light absorbed to the concentration of the absorbing molecule. At a wavelength of 260 nm, the average extinction coefficient for double-stranded DNA is 0.020 (μg/ml)^-1 cm^-1, for single-stranded DNA and RNA it is 0.027 (μg/ml)^-1 cm^-1 and for short single-stranded oligonucleotides it is dependent on the length and base composition. Thus, an optical density (or "OD") of 1 corresponds to a concentration of 50 μg/ml for double-stranded DNA. This method of calculation is valid for up to an OD of at least 2.

To determine the quantity and purity of extracted plasmid DNA,there are some step of calculation required.

DNA concentration (µg/mL):

= 50 µg/mL x OD260 x dilution factor

Dilution factor is the final volume / aliquot volume.aliquot volume is the measure of sub volume of original sample . final volume is the total volume.

dilution factor =final volume /aliquot vol.

The total yield in 500µL sample:

= DNA concentration x volume of sample in milliliters

The concentration of the plasmid is dependent on copy number and elution volume. If a higher concentration is desired for subsequent applications, perform an ethanol precipitation after plasmid isolation.

 RESULTS:

Photometry reading:

OD230 = 0.218

OD260 = 0.357

OD280 = 0.191

Ratio of (OD260/ OD280):

= (0.357 ÷ 0.191)

= 1.8691

Ratio of (OD260/ OD230):

= (0.357 ÷ 0.218)

= 1.6376

DNA concentration (µg/mL):

= 50 µg/mL x OD260 x dilution factor

= 50 x 0.357 x 50

= 892.5 µg/mL

Total yield in 500µL sample:

= DNA concentration x volume of sample in milliliters

= 892.5 µg/mL x 0.50 mL

= 446.25 µg

CONCLUSION:

 As a conclusion, with advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. During this experiment the student carry out the extraction of the plasmid DNA from plasmid-containing culture. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity.

lAB 5: DETERMINATION OF ANTIMICROBIAL EFFECTS OF MICROBIAL EXTRACTS

INTRODUCTION

 Certain group bacteria can produce antimicrobial substances with the capacity to inhibait the growth of pathogenic and spoilage micro-organism.An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic(s); today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic(s) has come to denote a broader range of antimicrobial compounds, including anti-fungal and other compounds. While antibiotic is A substance that is capable of stopping the growth of, or destroying, bacteria and other microorganisms which cause bacterial disease. Many antibiotics are themselves produced by microorganisms (bacteria and molds). 

Antibiotics are germicides that are safe enough to be swallowed or injected into the body. With advances in medicinal chemistry, most of today's antibacterials chemically are semisynthetic modifications of various natural compounds. These include, for example, the beta-lactam antibacterials, which include the penicillins (produced by fungi in the genus 'Penicillium'), the cephalosporins, and the carbapenems. Compounds that are still isolated from living organisms are the aminoglycosides, whereas other antibacterials—for example, the sulfonamides, the quinolones, and the oxazolidinones—are produced solely by chemical synthesis. Accordingly, many antibacterial compounds are classified on the basis of chemical/biosynthetic origin into natural, semisynthetic, and synthetic. Another classification system is based on biological activity; in this classification antibacterials are divided into two broad groups according to their biological effect on microorganisms: bactericidal agents kill bacteria, and bacteriostatic agents slow down or stall bacterial growth.

RESULT: 

 part 1

Strains of LAB	Strains of spoilage/pathogenic bacteria	Inhibition zone (cm)
LAB Species	Staphylococcus aureus	(1.4+1.3)/2 =1.35 cm
	Escherichia coli	(0.8+1.0)/2 =0.9 cm

part 2

E COLI

A	B
0.173	0.172
0.270	0.225
0.346	0.362
0.392	0.389

Figure 1: graph  shows serial dilution of extracellular extract using E.coli

 S.aureus

A	B
0.557	0.659
0.770	0.670
0.820	0.960
0.846	0.981

Figure 2 : Graph shows serial dilution of extracellular extract using S.aureus

DISCUSSION

Part  1. Determination of bacterion activity via agar diffusion test
 

The concentration of the compound will be highest next to the disk, and will decrease as distance from the disk increases. If the compound is effective against bacteria at a certain concentration, no colonies will grow where the concentration in the agar is greater than or equal to the effective concentration. This is the zone of inhibition. Thus, the size of the zone of inhibition is a measure of the compound's effectiveness: the larger the clear area around the filter disk, the more effective the compound.

Part  2. Determination of bacterion activity via optical density

There is one step that we need to prepare a negative-control for ‘auto-zero’ via spectrophotometer.

 

 Spectrophotometry involves the use of a spectrophotometer. A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.

Spectrophotometers are commonly used for the measurement of transmittance or reflectance of solutions, transparent or opaque solids, such as polished glass, or gases. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm - 2500nm using different controls and calibrations. Within these ranges of light, calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination.

An example of an experiment in which spectrophotometry is used is the determination of the equilibrium constant of a solution. A certain chemical reaction within a solution may occur in a forward and reverse direction where reactants form products and products break down into reactants. At some point, this chemical reaction will reach a point of balance called an equilibrium point. In order to determine the respective concentrations of reactants and products at this point, the light transmittance of the solution can be tested using spectrophotometry. The amount of light that passes through the solution is indicative of the concentration of certain chemicals that do not allow light to pass through.

The use of spectrophotometers spans various scientific fields, such as physics, materials science, chemistry, biochemistry, and molecular biology. They are widely used in many industries including semiconductors, laser and optical manufacturing, printing and forensic examination, as well in laboratories for the study of chemical substances. Ultimately, a spectrophotometer is able to determine, depending on the control or calibration, what substances are present in a target and exactly how much through calculations of observed wavelengths.

CONCLUSION 

 In conclusion, the results show that lactic acid bacteria(LAB) may act as a bio preservatives. From the experiment, the LAB succesfully shows its bio preservatives properties on both gram-positive and gram-negative bacteria which are Escherichia coli and Staphylococcus aureus. Antimicrobial compounds produced by LAB have provided these organisms with a competitive advantage over other microorganisms.

LAB 4: SOURCES OF CONTAMINATION AND INFECTION

INTRODUCTION:

What is Contamination?

Contamination is a process or act that causes materials or surfaces to be soiled with contaminating substances. There are two broad categories of surface contaminants: film type and particulates. These contaminants can produce a “killer defect” in a miniature circuit.  Film contaminants of only 10 nm (nanometers) can drastically reduce coating adhesion on a wafer or chip. It is widely accepted that particles of 0.5 microns or larger are the target. However, some industries are now targeting smaller particles.

 Sources of Contamination

This is a partial list of some of the commonly known contaminants that can cause problems in some cleanroom environments. It has been found that many of these contaminants are generated from five basic sources. The facilities, people, tools, fluids and the product being manufactured can all contribute to contamination.

What Is Infection?

 An invasion and multiplying of pathogenic microbes in the body tissues in which they are not usually present. Pathogenic means capable of causing disease. Infection does not always cause a disease.

Sources of infection

The source of  infection is the location from which an infection is acquired.

OBJECTIVE:

to determine the microorganism in the air and from healthy humans

RESULT:

We got the result after 24 hours.

Calculating microorganisms colony :

AIR:

1) 8

2) 15

HANDS:

1) 1454

2) 2536

EAR:

1) present

2) present

NORMAL BREATHING:

1) 5

2) 7

VIOLENT COUGHING:

1) 32

2) 7

DISCUSSION:

   Contamination is the presence of a minor and unwanted constituent (contaminant) in material, physical body, natural environment, at a workplace, many more.

    Airborne microorganism are usually carried on dust particles,although some (fungal spores) carried directly by air currents.

   Resident microorganisms (colonizing or endogenous flora) include

Staphylococcus species and diptheroids. These microorganisms are

considered permanent residents of the skin and are not readily

removed by mechanical friction. Resident microorganisms in the deep

layers may not be removed by handwashing with plain soaps and

detergents, but they can usually be killed or inhibited by hand hygiene

with products that contain antimicrobial ingredients.

    

      Transient microorganisms (non-colonizing or exogenous flora) include

microorganisms that come into contact with skin through interactions

with patients, with equipment, or with the environment. Non-colonizing

flora are not consistently present in the majority of persons and survive

only a limited period of time. These organisms are most frequently

associated with Hospital-acquired infections and are often acquired

through activities that involve close contact with a patient’s secretions

or excreta. Non-colonizing flora are easily removed by routine handwashing.

CONCLUSION:

Bacteria are an important group of living organisms. Most of them are microscopic and unicellular, with a relatively simple cell structure, lacking a cell nucleus, and organelles such as mitochondria and chloroplasts. During this experiment we can determine many type of microorganism that present in our whole body. For example airborne, resident and transient microorganism are all around us. As the conclusion, don’t eat or drink from unhygienic places. One should realize that while good bacteria can survive without us, we cannot survive without good bacteria.