A TRAINING REPORT
ON ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF BENEFICIAL MICROBES (BACTERIA, FUNGI AND ACTINOMYCETES) FROM SOIL AND VERMICOMPOST
Submitted to
Gautam budh technical University for Partial fulfillment for the award of the degree of Bachelor’s of Technology IN BIOTECHNOLOGY
2013 Under the guidance of
Dr. Biotechnology and Bioresource Division Centre For Mycorrhizal Research
The Energy And Resources Institute (TERI) Indian Habitat Centre, New Delhi Submitted By
SAURABH KUMAR GAUTAM B.Tech Biotechnology DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF ENGINEERING & TECHNOLOGY IILM ACADEMY OF HIGHER LEARNING GREATER NOIDA
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ISOLATION, IDENTIFICATION AND CHARACTERIZATION OF MICROCROORGANISMS (BACTERIA, FUNGI AND ACTINOMYCETES) FROM SOIL AND VERMICOMPOST
A TRAINING REPORT Submitted by
Saurabh kumar Gautam (0915054044) in partial fulfillment for the award of the degree of BACHELOR’S OF TECHNOLOGY in
Biotechnology
DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF ENGINEERING & TECHNOLOGY IILM ACADEMY OF HIGHER LEARNING GREATER NOIDA
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DECLARATION
I, Saurabh Kumar Gautam, student of Bachelor’s of Technology in Biotechnology (7th semester), College Of Engineering & Technology,Iilm Academy Of Higher Learning,Greater Noida, hereby, declare that the work presented in the training report entitled, " ISOLATION, PURIFICATION AND CHARACTERIZATION OF BENEFICIAL MICROBES (BACTERIA, FUNGI AND ACTINOMYTES) FROM SOIL AND VERMICOMPOST " is an authentic record of my work done from 20rd june 2012 to 20th august 2012, under the supervision of Dr. R. K. Mishra, reseacher, Center of Mycorrhizal Research, Biotechnology and Bioresource Division, The Energy And Resources Institute (TERI).
Saurabh Kumar Gautam (0915054044)
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CERTIFICATE
This is to certify that Saurabh Kumar Gautam has carried out his training work entitled " ISOLATION, PURIFICATION AND CHARACTERIZATION OF BENEFICIAL MICROBES (BACTERIA, FUNGI AND ACTINOMYTES) FROM SOIL AND VERMICOMPOST "
of Bachelor’s of Technology in Biotechnology (7th semester),
College Of Engineering & Technology,Iilm Academy Of Higher Learning,Greater Noida aryana from 19th june 2012 to 19th August 2012 in the Centre for Mycorrhizal Research, Biotechnology and Bioresource Division, TERI under my guidance and supervision.
Dr. Biotechnology and Bioresource Division Centre For Mycorrhizal Research The Energy And Resources Institute (TERI), New Delhi
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ACKNOWLEDGEMENT I humbly bow my head before lord almighty for blessing me with the power to complete this endeavor successfully. I express deep sentiments of gratitude to Dr. R.K.Pauchauri, Director General The Energy and Resources Institute (TERI), New Delhi, for granting me permission to work in this esteemed institute.
For me, it was great honor to work under with my guide Dr. R.K. Mishra,, Biotechnology and Bioresources Division & centre for Mycorrhiza Research, TERI New Delhi. I am very thankful to him for the precious advise, consistent encouragement and moral support, critical and fruitful indulgence despite of busy schedule during knotty hours during my work period and without whom I would have never been able to submit my work. I put my respects and honor for Dr. Alok Adholeya Director Biotechnology and Bioresources Division the granting work in his division I put on record the timely suggestion and guidance received from Dr. Reena Singh for eliciting novel, supportive and perspectives towards the whole approach of the project. I extend my sincere thanks to Dr. Sashidhar Burla, Dr. Manab Das, Dr.mandira and Mr. Sachin Rastogi for their valuable support. I am equally grateful to the CMR team: Ms Amrit Preet Kaur, Ms Rita, and Ms Poornima Saraswat and Ms yeti for their moral support and guidance which greatly contributed to the success of the present study. They had been very kind and patient while suggesting me the outlines of this project and correcting my doubts and nurturing a comfortable atmosphere. With deep respect, I express my sincere gratitude to Dr. Sonia Advani (Head of Department) and other faculty members), College Of Engineering & Technology, Iilm Academy Of Higher Learning , for their valuable suggestion and encouragement. I sincerely Thank Ms Nazz , Ms Aditi And Ms Charu Mr. Sandeep, Ms. Sreeparna, Ms. Amrit, Ms. Shilpi, Ms. Ankita, Ms. Rita, Mr. Pavan, Mr. Sachin, Mr Amit, and, Mr. Akhilesh Mr. Pavan Sunkireddy from the core of my heart. I also express my truthful thanks to Mr. Joshi. I would also like to make note of the immense support of Mr. Akhilesh I have no words to express the moral support shared with my friends during my course work. Thanks are also due to the entire management and staffs who have directly and indirectly helped me to complete my work. Last, but not least, I owe my gratitude to my parents and teachers for all the love and affection they have been showered upon me. Though mere words could not convey it all, still it is an attempt to express my heartfelt gratitude to everybody who made the project successful. I am submitting this project as a “TRAINEE HANDBOOK FOR BIOTECHNOLOGY� so that it becomes easier for new trainees and they can work in a better way.
Saurabh Kumar Gautam
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TRAINEE HANDBOOK FOR BIOTECHNOLOGY
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INDEX Index Chapter one…………Basic techniques………………………………….…8 Chapter two…..........Serial dilution……………………………………….16 Chapter three ……….Spreading………………………………………….23 Chapter four………..Incubation, counting and selection……………….26 Chapter five…………Streaking………………………………………….28 Chapter six…….........Agarose gel electrophoresis……………………….31 Chapter seven……….DNA isolation from fungi………………………...37 Chapter eight………...DNA isolation from bacteria……………………..41 Chapter nine…………PCR amplification………………………………..44 Chapter ten…………..PCR product purification………………………...47 Chapter eleven……….Dos and don’ts…………………………………...49 Chapter twelve ………Bioinformatics…………………………………...54 Example…………………………………………………………………..57 References………………………………………………………………...60
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CHAPTER 0NE BASIC TECHNIQUES
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B ASIC TECHNIQUES FOR MICROBIAL STUDIES
(These techniques can be used to grow bacteria, fungi, actinomycetes and can be modified according to the need) Microbiological media Microorganisms will grow on practically any source of organic food which provides carbon compounds used for energy,, and nitrogen compounds to be incorporated into proteins for growth. These substances are normally present dissolved in water. er. However, in nature, microorganism (bacteria) can break down solid and insoluble substances by releasing enzymes into the substrate in which they are growing. These substances are thus broken down or digested to simpler substances and the process is called extracellular digestion because it takes place outside the cells. The four types of media are used in microbiological studies are – Nutrient Broth, PDB (Potato Dextrose Broth) B usually in tubes Nutrient Agar and PDA (Potato Dextrose Agar),, which is set into a jelly by the addition of a seaweed extract called agar, and when melted poured into glass or plastic Petri dishes - also known as "plates" "plates".
Figure1 igure1: For bacteria- broth
Figure 2: 2 Proper use of petriplates
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A standard carbon source is glucose, and nitrogen is often provided by peptones (partially digested proteins), or inorganic salts. Minerals and vitamins may also be provided, according to the growth requirements of the bacteria. Combinations of chemicals (buffers) may be used to keep the pH stable. Measured amounts of the concentrates are added to water, and dissolved to reconstitute the media. NOTE:
Use clean spatula for weighing different components. Do not keep containers of content open for long to prevent moisture from going inside. Never prepare media up to the quantity of the bottle or jar example never prepare 1L media in 1L bottle because during autoclaving due to pressure it may burst and its tough during the pouring process.
Sometimes, substances are mixed into media, in order to suppress growth of other types of bacteria. There are many such selective media Note :( antibiotics are added in PDA plates to prevent bacterial contamination in fungi) NA (doesn’t need antifungal because the NA only supports bacterial growth.) Microbiological techniques
Microbiological Basic Techniques
Sterilisation
Aseptic Techniques
Inoculation
Incubation
Figure 3: Basic techniques Sterilisation These media must then be sterilised by heating in an autoclave (like a pressure cooker) at 121°C (pressure 1 bar or 15 lb/sq. in.) for 15 minutes, which kills all living organisms, including spores. All apparatus used from this point onwards must be sterilised by heat (glassware - 160 °C for 2 hrs) or exposure to radiation (oven used for drying can do this work)
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Aseptic techniques Must be used to reduce the likelihood of bacterial/fungal/other microorganism’s contamination. This usually involves disinfection of working areas by wiping it with the 70% ethanol (spirit), minimising possible access by bacteria from the air to exposed media, and use of flames to kill contamination which might enter vessels as they are opened. Note: the more the clean work area you keep the less chances of contamination are there
Figure 4: pouring a plate Pouring a plate: The pouring a plate should be done very carefully in the laminar hood to prevent any contamination seeping in from hand and the mouth of the jar should be sterilized before pouring.
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Inoculation Inoculation of Bacteria, fungi and actinomycetes may be done by exposing them to the media (inoculated) by various means. Usually the for bacteria from a drop in a heat-sterilised loop are spread on the surface of (ready set) agar. A similar technique is used with broth cultures. We can pick up a single mycelia or spore of the fungi using a sterilized needle for inoculation. Same we can do for actinomycetes. We can even use a micro tip for inoculation of single isolated colony from the bacteria.
VIDEO TUTORIAL http://www.youtube.com/watch?v=X5dU98-K5_g&feature=related http://www.youtube.com/watch?v=PiWwnBbCrNs&feature=related
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Figure 5: Inoculation
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Figure 6: Sealing a plate
Sealing is done by wrapping the sealer around the circumference plate or sealing as shown in the figure Sometimes bacteria in a liquid are introduced using a sterile pipette to the Petri dish before the (fairly cool) agar medium is poured on top ("pour plates"). Then the Petri dishes containing agar or tubes containing broth are incubated, i.e. put in a special apparatus at a fixed temperature (usually 37°C - human body temperature, for possible pathogens - or 25°C for bacteria from the environment and fungi at 26ºC). In schools, lower incubation temperatures are used in order to discourage the growth of potential pathogens. NOTE:
When growing bacteria, it is usual to invert the Petri dishes, so as to prevent condensation droplets from falling onto the surface of the agar. Petri dishes are often "sealed" at this stage to prevent people who handle them from contamination by bacteria, which will multiply greatly. It is normal to use 2 strips of adhesive tape from base to lid rather than attempt seal the circular edge of the Petri dish. This is to guard against the possibility of anaerobic organisms growing due to lack of air. However, it must be borne in mind that any drips from a partially sealed Petri dish are potential sources of infection.
Figure 7: Storing a plate in incubator
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Results Cultures are usually examined after 24 hrs incubation. Bacteria generally grow in 24-48 hours. Whereas the fungi can take 3 to 5 days for spores to appear depending on their growth rate. Liquid media such as broth become cloudy if bacteria are present. This could be the result of only one bacterial cell originally entering the medium, then dividing repeatedly to produce Millions!
Figure 8: Broth after incubation Bacteria on agar "plates" become visible as distinct circular colonies; each colony should represent an individual bacterial cell (or group) which has divided repeatedly but, being kept in one place, the resulting cells have accumulated to form a visible patch.
Figure 9:Different types of organisms which can come in plate By an extension of this method using serial dilutions in sterilised liquids, the number of bacteria in a given amount of sample, e.g. food, can be calculated. After use, cultures, etc. must be sterilised before discarding.
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CHAPTER 2 SERIAL DILUTION
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SERIAL DILUTION It is a common method to determine microbial density and diversity for both liquid and solid samples . Most samples (soil) have high enough numbers of microorganisms that has to be serially diluted to quantities effectively. The following is a step-by-step procedure for dilution , and includes some practice problems at the end. This can help us to find bacterial, fungal, or viral counts. This protocol is specific for bacterial counts (colony-forming units, CFUs), but can be modified for fungi (CFUs) and viruses (plaque-forming units, PFUs for viral counts). History Robert Koch is credited with identifying a method for bacterial enumeration, used first for the study of water quality. His article, About Detection Methods for Microorganisms in Water. Was published in 1883. The standard plate count is a reliable method for enumerating bacteria, fung and actinomycetes . A set of serial dilutions is made, a sample of each is placed into a liquefied agar medium, and the medium poured into a Petri dish. The agar solidifies, with the bacterial cells locked inside of the agar. Colonies grow within the agar, as well as on top of the agar and below the agar (between the agar and the lower dish). The procedure described above produces a set of pour plates from many dilutions, but spread plates (sample spread on top of solidified agar) can be used also. The agar plate allows accurate counting of the microorganisms, resulting from the equal distribution across the agar plate. This cannot be done with a fluid solution since 1) One cannot identify purity of the specimen, and 2) There is no way to enumerate the cells in a liquid.
Figure 10: colonies
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Figure 11: Serial dilution technique
THE STANDARD FORMULA
________________ Colony count (CFUs) on an agar plate_____________ Total dilution of tube (used to make plate for colony count) X volume plated Note: if solid sample the per gram Values needed: 
A colony count from the pour or spread plates,

A dilution factor for the dilution tube from which the countable agar plate comes, and
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
The volume of the dilution that was plated on the agar plate.
PROTOCOL STEP 1: Determine the appropriate plate for counting: Look at all plates and find the one with 30-300 colonies (see COMMENTS & TIPS section at end for explanation). Use the total dilution for the tube from where the plate count was obtained. If duplicate plates (with same amount plated) have been made from one dilution, average the counts together. STEP 2: Determine the total dilution for the dilution tubes: Dilution factor = amount of specimen transferred divided by the total volume after transfer [amount of specimen transferred + amount of diluent already in tube]. Determine the dilution factor for each tube in the dilution series. Multiply the individual dilution factor for the tube and all previous tubes. To calculate this dilution series:
Determine the dilution factor of each tube in the set. Dilution factor for a tube = _____amount of sample________ volume of specimen transferred + volume of diluent in tube But after the first tube, each tube is a dilution of the previous dilution tube. SO‌.. total dilution factor = previous dilution factor of tube X dilution of next tube FOR THE ABOVE DILUTION SERIES: 0.5 ml added to 4.5ml = 0.5/5.0 = 5/50 = 1/10 for 1st tube 1ml added to 9ml = 1/10 (2nd tube) X previous dilution of 1/10 (1st tube) = total dilution of 1/100 for 2nd tube.
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STEP 3: Determine the amount plated (the amount of dilution used to make the particular pour plate or spread plate). There is nothing to calculate here: the value will be stated in the procedure, or it will be given in the problem. STEP 4: Solve the problem
1. The countable plate is the one with 51 colonies. 2. The total dilution of the 2nd tube from which that pour plate was made = 1/102 3. The amount used to make that pour plate = 0.1ml (convert to 1/10 - it is easier to multiply fractions and decimals together). 51 colonies = 51 X 103 = 5.1 X 104 (scientific notation) OR 51,000 CFUs/ml 1/102 X 1/10 45 colonies = 45 X 104 = 4.5 X 105 (scientific notation) OR 450,000/ml 1/103 X 1/10
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Safety Tubes and agar plates should be discarded properly in a biohazard container for proper sterilization. The pipettes will also be sterilized (washed first if using reusable glass pipettes. Do not pipette by mouth. Use sterile technique in the transfer of microorganisms from tube to tube, as well as in the production of the pour plates. . Comments and Tips: Large number of colonies on the agar plate and very less than leads to a high degree of error. Air contaminants can contribute significantly to a really low count. A high count can be confounded by error in counting too many small colonies, or difficulty in counting overlapping colonies. Use sterile pipettes or tips for the dilutions, and use different ones in between the different dilutions. To do otherwise will increase the chances of inaccuracy because of carry-over of cells. Accuracy in quantisation is determined by accurate pipette use and adequate shaking or vortex of dilution tubes/eppendofs.
VIDEO TUTORIAL http://www.youtube.com/watch?v=NXirIFfpKdg http://www.youtube.com/watch?v=l8HgDPajsMI&feature=related
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CHAPTER THREE SPREADING
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SPREADING
One method of spreading bacteria/fungi evenly over the surface of an agar plate medium is commonly referred to as the spread plate method. Classically a small volume of a bacterial suspension is spread evenly over the agar surface using a sterile bent glass rod as the spreading device. This evenly distributing the (sample) suspension is typically to permit the growth of colonies that can subsequently be enumerated. Each plate is spread with a single inoculum of the l suspension. An alternative approach to spreading a single inoculum volume with a smooth device is to apply a smaller volume and tip the plate, allowing gravity (by tilting the plate) to distribute the inoculum in a band or track (track method) or to allow the inoculum to dry in place (drop method). With this alternative approach, several sample dilutions can be distributed on a single agar plate. History: Since the development of the agar plate in Robert Koch's laboratory, several methods have been used to achieve an even distribution of bacterial growth on or in the agar. The most common methods used to achieve this type of distribution are: spread, pour, thin-layer, layered, and membrane filter Principles: Using the spread method a small volume of a bacterial/fungal (sample) suspension is distributed evenly over the surface of an agar plate using a smooth sterilized spreader (by dipping in spirit and flaming it). In the case of track plates, gravity is used to spread the inoculum down the agar in a column forming a track Spread Plates Agar plates: Select and prepare an agar medium based upon the type of bacteria to be enumerated or selected. After autoclaving, cool the agar to between 45°C and 50°C prior to pouring the plates to minimize the amount of condensation that forms. The thickness of the agar should be roughly 0.3 cm, which can be achieved by pouring 15 to 20 ml of media per 100 x 15 mm plate. (Marking is generally there in the plate or half of the plate has to be purified) NOTE:
Freshly prepared plates do not work as well as dry plates as it takes longer for the inoculum to absorb into the agar. Plates may be dried by keeping them at room temperature for roughly 24 hours. Plates will dry faster in lower humidity so placing them in a laminar flow hood will speed up the drying process.
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
Once dried, plates may be used or refrigerated in closed bags or containers until required. Refrigerated plates should be warmed to room temperature prior to use. Inoculations: When enumerating colony-forming units (CFUs), CFUs can be used to calculate the number of CFUs/ml of the original sample. Typically a dilution series is prepared, often a ten-fold dilution series, using a suitable dilutent such as phosphate-buffered saline.
Serial Dilution Protocols a convenient inoculum volume, in terms of spreading, absorption, and calculations, is 0.1 ml (100 microliters). Since some microorganism (bacteria) rapidly attach to the agar surface, the inoculum should be spread soon after it is applied. Working from the most dilute suspension to the most concentrated is advised. Starting from most dilute to most concentrate makes it unnecessary to change pipette tips between the dilutions. Spreading: A reusable glass(this is mostly used) or metal spreader should be flame sterilized by dipping in alcohol (such as 70% isopropyl or ethanol), shaking off the excess alcohol, and igniting the residue .The spreader is then allowed to cool. The spreader is placed in contact with the inoculum on the surface of the plate and positioned to allow the inoculum to run evenly along the length of the spreader. Even pressure is applied to the spreader and the plate is spun, on a turntable or by hand. Alternatively the spreader may be rotated over the agar surface Avoid spreading the inoculum all the way to the edge of the agar. The goal is to evenly distribute the inoculum and to allow it to be absorbed into the agar. The plate, or spreader, should be rotated long enough to avoid pooling along the spreader once the rotation is stopped. Never spread the inoculum in a zig zag manner which will never give uniform colonies. Avoid disturbing plates for 10 to 20 minutes after spreading. Drying time varies with the room temperature and humidity.
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CHAPTER FOUR INCUBATION, COUNTING AND SELECTION
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Incubation: After the spread plates have absorbed the inocula for 10 to 20 minutes they may be inverted and incubated as desired. Observe the plates before the colonies have had time to fully develop. Closely positioned colonies may be difficult to resolve as separate colonies later. Continue the incubation as necessary. Incubation in closed humidified containers will help avoid problems with plates drying out when working with slow-growing colonies. Counting and Selection: After appropriate incubation, plates are inspected. When plating a dilution series, the growth on the plates should reflect the predictable drop in CFUs/plate as illustrated in this picture of a 10-fold dilution series prepared from an overnight culture.
a)
b)
Figure 11: colonies at dilution -4 and colonies at dilution -6.
Plates with well isolated colonies may be inspected and, if desired, colonies "picked" to establish new cultures for purification.
VIDEO TUTORIAL:
http://www.youtube.com/watch?v=AaG3Pt3nwLQ
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CHAPTER FIVE STREAKING
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STREAKING (Used for bacterial purification) Purpose The streaking procedure is used in order to check 1) Whether a culture is a "pure culture� or if there are more than one organisms in it (contaminated) 2) Whether a culture is viable, i.e. able to grow (to check a "stock" culture kept in the fridge for storage) Background The basic tools used in this procedure include a “loop"(can be replaced by sterile micro pipette tip), and a Petri dish containing an appropriate media agar, well cooled and set, with a dry surface. The principle is that individual microbial cells can be separated by dragging them over the surface of the agar, and then given a chance to grow into individual colonies Materials Petri plate containing (set - fairly firm -dry) Nutrient Agar. Wire loops, mounted in metal handle Inoculum material in broth or other (liquid) medium Procedure Clear an area of bench, and wipe it with disinfectant (ethanol). Flame a wire loop, bringing it all to red heat, and leave it upright in a rack to cool. (Optional - repeat to get several cooled loops)
1) Using the loop, take a drop of the liquid culture medium (broth) provided and spread it carefully in a line across the surface of the agar as shown. With the same loop, a second, third and fourth line may be drawn parallel to the first. Close the lid of the Petri dish immediately. 2) Sterilise the loop in the flame once again, and allow it to cool. 3) turn the Petri plate so that the end of the previous lines can be the start of the next ones. 4) Take a cooled loop and make 2 or 3 strokes as before. Close the lid of the Petri dish immediately. Repeat 2, 3, 4 until there is no more space round the edge (4 or 5 times), then finish off with a single zigzag streak across the middle.
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Seal and label the Petri plate with the culture reference and your name and the date. Place it in an inverted position in the incubator at an appropriate temperature.
VIDEO TUTORIAL http://www.youtube.com/watch?v=Ay2hhujTuvg&feature=related http://www.youtube.com/watch?v=_1KP9zOtjXk http://www.sumanasinc.com/webcontent/animations/content/streakplate.html
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CHAPTER SIX AGAROSE GEL ELECTROPHORESIS
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AGAROSE GEL ELECTROPHORESIS
Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA. We will be using agarose gel electrophoresis to determine the presence and size of PCR products.
Background: Electrophoresis is a method of separating substances based on the rate of movement while under the influence of an electric field. Agarose is a polysaccharide purified from seaweed. An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the solution becomes clear, and then pouring it into a casting tray and allowing it to cool. The result is a flexible gelatin-like slab. During electrophoresis, the gel is submersed in a chamber containing a buffer solution and a positive and negative electrode. The DNA to be analyzed is forced through the pores of the gel by the electrical current. Under an electrical field, DNA will move to the positive electrode (red) and away from the negative electrode (black) [we tend to get most confused in this context]. Factors’ influencing the DNA movement includes;
the strength of the electrical field, the concentration of agarose in the gel and most importantly, The size of the DNA molecules.
Smaller DNA molecules move through the agarose faster than larger molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized by the use of a dye that binds to DNA.
Purpose: To determine the presence or absence of PCR products and quantify the size (length of the DNA molecule) of the product.
Materials needed:
Agarose TAE Buffer 6X Sample Loading Buffer DNA ladder standard Electrophoresis chamber Power supply Gel casting tray and combs
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DNA stain Staining tray Gloves Pipette and tips
Recipes:
TAE Buffer
(now ready made stock solutions are available)
4.84 g Tris Base 1.14 ml Glacial Acetic Acid 2 ml 0.5M EDTA (pH 8.0) - bring the total volume up to 1L with water
Add Tris base to ~900 ml H2O. Add acetic acid and EDTA to solution and mix. Pour mixture into 1 L graduated cylinder and add H2O to a total volume of 1 L. Note – for convenience a concentrated stock of TAE buffer (either 10X or 50X) is often made ahead of time and diluted with water to 1X concentration prior to use.
6X Sample Loading Buffer 1 ml sterile H2O 1 ml Glycerol Enough bromophenol blue to make the buffer deep blue (~ 0.05 mg) EtBr is added to visualize the DNA under UV now a visualization machine can be used to visualize and record the DNA run. But be very careful using EtBr its carcogenic. -for long term storage, keep sample loading buffer frozen.
QUIKView DNA Stain 25 ml WARDS QUIKView DNA Stain 475 ml warm water (50-55ď‚° C)
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Agarose Gel Electrophoresis Protocol
Preparing the agarose gel • Measure 1.5 g Agarose powder and add it to a 500 ml flask • Add 150 ml TAE Buffer to the flask. (The total gel volume well vary depending on the size of the casting tray) • Melt the agarose in a microwave or hot water bath until the solution becomes clear. (If using a microwave, heat the solution for several short intervals - do not let the solution boil for long periods as it may boil out of the flask). • Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. • Seal the ends of the casting tray with two layers of tape. • Place the combs in the gel casting tray. • Pour the melted agarose solution into the casting tray and let cool until it is solid (it should appear milky white). • Carefully pull out the combs and remove the tape. • Place the gel in the electrophoresis chamber. • Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel. Note – gels can be made several days prior to use and sealed in plastic wrap (without combs). If the gel becomes excessively dry, allow it to rehydrate in the buffer within the gel box for a few minutes prior to loading samples.
Loading the gel • Add 6 l of 6X Sample Loading Buffer to each 25 l PCR reaction/ available purified DNA for demo purpose • Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder9very important write and write whatever you do. • Carefully pipette 20 l of each sample/Sample Loading Buffer mixture into separate wells in the gel. • Pipette 10 l of the DNA ladder standard into at least one well of each row on the gel. Note – if you are running multiple gels, avoid later confusion by loading the DNA ladder in different lanes on each gel.
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Running the gel
• Place the lid on the gel box, connecting the electrodes. • Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”) • Turn on the power supply to about 100-120 volts. Maximum allowed voltage will vary depending on the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes! (Not may know this) • make sure the current is running through the buffer by looking for bubbles forming on each electrode. • Check to make sure that the current is running in the correct direction by observing the movement of the blue loading dye(I made a mistake long back) – this will take a couple of minutes (it will run in the same direction as the DNA). • Let the power run until the blue dye approaches the end of the gel. • Turn off the power. • Disconnect the wires from the power supply. • Remove the lid of the electrophoresis chamber. • Using gloves, carefully remove the tray and gel.
Gel Staining (NOT REQUIRED THESE DAYS VISUALISATION AND RECORDING MACHINES ARE AVAILABLE)
• Using gloves remove the gel from the casting tray and place into the staining dish. • Add warmed (50-55°) staining mix. • Allow gel to stain for at least 25-30 minutes (the entire gel will become dark blue). • Pour off the stain (the stain can be saved for future use).
• Rinse the gel and staining tray with water to remove residual stain. • Fill the tray with warm tap water (50-55°). Change the water several times as it turns blue. Gradually the gel will become lighter, leaving only dark blue DNA bands. Destain completely overnight for best results. • View the gel against a white light box or bright surface.
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• Record the data while the gel is fresh, very light bands may be difficult to see with time.
VIDEO TUTORIAL http://www.youtube.com/watch?v=wXiiTW3pflM&feature=related https://www.youtube.com/watch?v=6mQGNDnOyH8
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CHAPTER SEVEN DNA ISOLATION FROM FUNGI
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Isolation of genomic DNA (As given in kit it can be used for various cells like fungus, plant including algae) DNA isolation is carried out using the DNeasy Plant Mini Kit(the protocol are available with the kits the principle remains same follow strictly according to manufacturer read them nicely and then follow them strictly) according to the manufacturer’s protocol. The procedure includes cell lysis, digestion of RNA by RNAse A, removing of precipitates and cell debris, DNA shearing, DNA precipitation and purification.
cell lysis digestion of RNA removal of precipitates and cell debris DNA shearing DNA precipitation purification
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Protocol for genomic DNA isolation: Around 100-300mg of fungal mycelia is taken in an eppendorf tube. The fungal mycelia should be washed with autoclave distilled water around 5-6 times and at last centrifuge the sample at 140000rpm for 5 minutes to remove the excess media. (Fungus can be grown in broth or can be scrapped from the agar plate)
The sample is taken in mortar and grind into fine powder using liquid N2 with pestle. 400µl of AP1 buffer is added to it and put the sample to thaw. The sample is then transferred into 2 ml of eppendorf tube and 4µl of RNase A is added to it & vortex vigorously. The sample mixture is incubated for 10 minutes at 65o. After that 130µl of AP2 buffer is added to the above and incubate for 5 minutes on ice. The lysate is centrifuge for 5 minutes at 14000rpm. The supernatant is pipit into the QIAshredder Mini spin column (lilac) placed in a 2 ml collection tube, and centrifuged for 2 min at 20,000 x g (14,000 rpm). The flow-through fraction from above step is transferred into a new tube without disturbing the cell-debris pellet.(using pipette) 1.5 volumes of Buffer AP3/E are added to the cleared lysate, and mix by pipetting. (450 μl lysate, 675 μl Buffer AP3/E is added. Reduce the amount of Buffer AP3/E accordingly if the volume of lysate was smaller) 650 μl of the mixture from above is pipit, including any precipitate that may have formed, into the DNeasy Mini spin column placed in a 2 ml collection tube and centrifuged for 1 min at 8000 rpm and the flow-through is discarded. The above step is repeated with remaining sample and flow-through from collection tube is discarded. The DNeasy Mini spin column is placed into a new 2 ml collection tube, 500 μl Buffer AW is added and centrifuged for 1 min at 8000 rpm. The flow-through is discarded and reuses the collection tube in next step. 500 μl Buffer AW is added to the DNeasy Mini spin column, and centrifuged for 2 min at 14,000 rpm to dry the membrane. The DNeasy Mini spin column is transferred to a 1.5 ml micro centrifuge tube, and 100 μl Buffer AE is directly pipet onto the DNeasy membrane and incubated for 5 min at room temperature (15–25°C), & then centrifuged for 1 min at 8000 rpm to
39
elute. (Elution with 50 μl (instead of 100 μl) increases the final DNA concentration in the eluate significantly).
Quantification of isolated DNA The DNA yields and quality were assessed by standard electrophoresis through a 1% (w/v) gel red-stained agarose gel.
Protocol for agarose gel electrophoresis 1 gm of agarose was weighed. 1 X TAE buffer was made from 50X TAE buffer. 495 ml of Distilled water + 10 ml of 50 X TAE buffer i.e. 500ml of 1 X TAE buffer. Agarose was dissolved in 100 ml of 1 X TAE buffer. The solution was warmed in microwave till agarose dissolved in buffer. Cool for some time such that temperature comes to 60oC. 10- 15 µl of gel red was added in it. The solution was poured in the gel casting plate and left it undisturbed to solidify. After the gel was solidified, the comb was taken out from the gel. Then the plate was placed in an electrophoretic chamber and the 1 X TAE buffer was poured in electrophoretic chamber over the level of gel. 5µl of DNA sample and 1µl of loading dye were mixed and loaded in the wells of gel. 100 bp ladder was loaded in one of the well. The gel was run at 110V for 45min.
(For further refer to chapter on agarose gel electrophoresis)
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CHAPTER EIGHT DNA ISOLATION FROM BACTERIA
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STEPS BEFORE ISOLATION
Pure colony is picked and inoculated into broth(Nutrient Broth etc) Put on shaker for overnight growth at 140-150 rpm at 30 to 33 Transfer it into 1.5ml eppendof Genomic DNA isolation Note absolute ethanol is added to wash buffer prior to the initial use
Requirement:
Microcentrifudge tube, absolute ethanol, RNase
Step1 Cell harvesting/prelysis
Transfer cultured bacterial cells (1*109) to a 1.5 ml microcentrifudge tubes. Centrifuge for 1 minute at full speed and discard the supernatant. Add 200200µl µl of GT buffer to the tube and resuspend the cell pellet by vortex or pipetting Incubate at room temperature for 5 minutes. Proceed with lysis step
Step 2 Cell Lysis
Add 20 µl of Proteinase K to the sample mixture and mix by vortex. Incubate at 60ºC for 30-60 minutes to lyse the sample until the tissue particulates have dissolved. During incubation, invert the tube every 5 minutes.
Optional Step: RNA Degradation
If RNA-free genomic DNA is required, perform this optional step. Add 5 µl of RNase A (10 mg/ml) to the sample lysate and mix by vortex. Incubate at room temperature for 5 minutes.
Step 3 Protein Removal
Add 100 µl of Protein Removal Buffer to the sample lysate and vortex immediately for 10 seconds. Incubate on ice for 5 minutes. Centrifuge at 14-16,000 x g for 3 minutes.
Step 4
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DNA Precipitation
Transfer the supernatant from Step 3 to a clean 1.5 ml microcentrifudge tube. Add 300 µl of isopropanol and mix well by inverting. Centrifuge at 14-16,000 x g for 5 minutes. Discard the supernatant and add 300 µl of 70% ethanol to wash the pellet. Centrifuge at 14-16,000 x g for 3 minutes. Discard the supernatant and air-dry the pellet for 10 minutes.
Step 5 DNA Re-hydration
Add 50-100 µl of TE or ddH2O and incubate at 60ºC for 30-60 minutes to dissolve the DNA pellet. During Incubation, tap the bottom of tube to promote DNA re-hydration.
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CHAPTER NINE PCR AMPLIFICATION
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PCR Amplification of rDNA A ̴ 650bps of region of the ITS1-5.8S-ITS2 region of the rDNA of the fungus is amplified using ITS1 (5’–TCC GTA GGT GAA CCT GCG G-3’), and ITS4 (5’–TCC TCC GCT TAT TGA TAT GC-3’) primers. Polymerase chain reaction was carried using the thermo stable enzyme Taq Polymerase( 5U/µl) in 25 µl reaction mixture containing 1X PCR buffer, 25mM MgCl2, mM dNTPs, 5pM each of primers and 30ng of the fungal genomic DNA as template. The PCR was standardized to get maximum amplification with least non-specificity. A negative control reaction (without DNA template) was also set up. PCR conditions are given in Table 1. The reaction was stopped by chilling the mixture at 4oC.
PCR Reaction Mixture (20µl) Genomic DNA
2μl,
PCR Buffer
2μl
MgCl2 -
1.2μl,
Deoxyribonucleosidetriphosphates (dNTPs) -
0.4μl,
Forward primers
1μl
Reverse primers
1μl
universal for Fungus
Distilled water -
12.2μl, and
Taq DNA polymerase -
0.2μl
TABLE: PCR Condition for amplification PRIMER SEQUENCE(5’
3’)
PCR PROFILE
PAIRS 95oC; 5 min
PRODUCT PRODUCT SIZE
1
95oC; 1 min 55oC; 1 min
ITS1
TCCGTAGGTGAACCTGCGG
ITS4
TCCTCCGCTTATTGATATGC 72oC; 1 min 72oC; 1 min
̴ 650bps 30
ITS1-5.8SITS2 rDNA
1
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Quantification of amplified ITS bands was done by using agarose gel electrophoresis 5μl of the amplified PCR product was used for electrophoresis using 1.0% agarose gel in 1X TAE buffer. The gel was run at 110V for 45 min, using 1X TAE as running buffer. The gel was observed under Gel Doc.
Purification of PCR amplified product 2 μl of the Purified PCR product was checked on electrophoresis using 1.0% agarose gel in 1X TBE buffer The gel was run for 45 min at 110V, using 1X TBE as running buffer. The gel was observed under UV illumination to check the purity of DNA.
VIDEO TUTORIAL http://www.youtube.com/watch?v=buxwFXEpdTU
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CHAPTER TEN PCR PRODUCT PURIFICATION
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Protocol
QIAquick® PCR Purification Kit was used in my case.
1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the colour of The mixture is orange or violet; add 10 μl 3 M sodium acetate, pH 5.0, and mix. The colour of the mixture will turn yellow. 2. Place a QIAquick column (it’s a Colum with small filterate which is now available with most kits) in provided 2 ml collection tube 3. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s or apply vacuum to the manifold until all the samples have passed through the column. Discard flow-through and place the QIAquick column Back in the same tube.
Sample & Assay Technologies 4. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s or apply vacuum. S Discard flow-through and place the QIAquick Column back in the same tube. 5. Centrifuge the QIAquick column once more in the provided 2 ml collection tube For 1 min to remove residual wash buffer. 6. Place each QIAquick column in a clean 1.5 ml microcentrifudge tube. 7. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0– 8. to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 μl elution buffer to the centerOf the QIAquick membrane, let the column stand for 1 min, and then centrifuge. 8. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
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CHAPTER ELEVEN DOS AND DONTS
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During a Lab SAFETY DO's:
SAFETY DON'T's:
Keep your workbench neat and organized.
Place cultures near the edge of the bench.
Label all cultures/containers.
Mix unknown chemicals.
Read Manuals SOPS present in lab to Start working without doing through obtain information. background study. Ask how to discard used cultures.
Pour any live culture down the sink drain.
Wear your safety glasses at all times.
Take off your safety glasses or touch your face with soiled latex gloves.
Report accidents to the instructor immediately.
Attempt to clean up a spill by yourself or leave the lab to treat an injury by you.
Take a rest break now and then.
Be in a rush to finish.
Keep away the spirit bottle
Keep your hands wiped with spirit near flame.
KEEP YOURSELF AND OTHERS SAFE
Most Important Lab Safety Rules Hazards exist in the lab for toxic chemicals and microorganisms. Microbes, in particular have a great versatility to grow and proliferate in different environments (i.e. pathogens), and thus it is important that you conduct yourself safely at all times when in the laboratory. If the lab instructor becomes concerned for your safety, or the safety of others, Don’t get hurt or feel bad because it’s for your safely. 1. Note the location for the emergency exits, first aid kit, fire extinguishers, hand wash station, emergency showers, and eye wash apparatus. 2. Do not smoke, eat or drink in the laboratory, or place any object on near your mouth. Keep your books, laboratory manual and workbook at a reasonable distance from your work area. 3. If a fire starts, or the fire alarm sounds, unplug any electrical apparatus and vacate the laboratory in an orderly manner. 4. Practice good aseptic techniques by performing the following before starting each class:
Long hair should be tied back. Wear closed footwear to protect the feet. Wear a clean lab coat. Wear protective glasses at all times (if required). Wash your hands thoroughly with soap and water before starting your exercises and then use spirit to kill any microorganism if it’s there.
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Wear disposable latex gloves when handling products (e.g. whole blood, plasma, serum, or carcinogenic substances etc.).
5. Don’t place any hazardous or infectious materials in the sink. Do not dispose of any solid material in the sink (it blocks the drainage system and then total repair is required). 6. To clean up spills of microbial cultures, first containing the spill by placing a paper towel/tissue paper soaked in 70% ethanol over the spill area. Keep the towel on the spill for 20 minutes. Inform your instructor of the spill. Place the towel in an autoclave (biohazard) waste bag provided. Ensure you wash your hands immediately after dealing with the spill. 7. Never place any instruments or materials into your mouth. I.e. Do not pipette by mouth. Use the mechanical apparatus provided. 8. Place pipettes that are used during class immediately into the appropriate waste container. 9. Place used glass slides and cover slips in glass dishes of disinfectant. Do not discard any demonstration slides. 10. Place all used bottles, tubes and cultures in the containers provided on each bench for staff to remove and autoclave. 11. Place contaminated waste in the autoclave bag-lined containers. All other (nonbiohazard waste) can be disposed of in appropriately labeled containers. 12. All materials requiring incubation or refrigeration must be appropriately labeled and placed on the trays provided. 13. At the conclusion of each class clean your work bench. 14. Decontaminate your work bench at the end of each exercise by applying an antiseptic wash. 15. Before leaving the laboratory for a coffee, lunch a nature break, or at the end of the day, wash your hands thoroughly with antiseptic soap and water.
Some more dos and don’ts we should from time to time...... • Take a few minutes to clean up after you have worked in the lab. • All bottles must be labelled with at least: initials, contents, and date (w/year). This is a protocol followed by all functional and productive laboratories. Unlabeled bottles, including media, are discarded. • If a biohazard bag is full, seal it and place in an autoclave bin. Place new bag on rack. • Be sure to close the door fridge tightly when leaving. • Do not mix glassware with plastic products in the autoclave bins. The plastic melts i.e. only use autoclavable plastics. • When adding more materials to the autoclave-bound cart, don't just throw your stuff on the cart. There are autoclave bins on the cart. Place your items in an appropriate dust bin. • Wash your hands before leaving the lab.(for your and others safety) • Make what you need for the near future. • Prepared Petri plates are fine at room temperature for a good while. Refrigeration leads to condensation problems(less is more do slowly on time I used to make bulk plates then used to fight condensation).
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• • • • • • • • • • • • • • • • • • • • • •
• • • • • •
Do not use the PC while wearing gloves. All contaminated materials must be set in the autoclave bins next to the laminar flow hood. If you move equipment or supplies, be sure to return them to their original location before you leave. (this even helps you in life to be organised) When playing music in the lab, close the door to the hall. Sound carries. After using the PC, close the applications when finished. The PC next to the plate reader is to be used only for plate reader work. If you open a window to the outside, close it before leaving. Label your tip boxes and return them to your lab bench after use and fill them for further autoclaving. Remove tubes from the shaker and plates from the incubator when no longer needed. If using the gel documentation system, always check to be sure that you turned off the UV transilluminator. When using the Bunsen burner at the hood, double check to make sure you turned it off. If you're having a bad day, it's best to stay out of the lab. (Do some write work or use internet otherwise it becomes useless work divert yourself.) Better safe than sorry. Keep a thorough notebook and don't forget to maintain a useful table of contents.(learned from my guide Mishra sir in the best way thanks a lot sir) Be pleasant and supportive; everyone has a lot to do...not just you. If you don't know how to use a piece of equipment, ask someone who does. Don't experiment. After pouring plates, immediately rinse empty bottle with warm water. Otherwise a thin film remains after washing. If an item (e.g., tubes, tips, and media) is getting low, write it on the board and include your initials and the date. Do not remove or use anything from another person's lab bench/shelf without their permission. If media or agarose overflows in the microwave, clean it up while it is still in liquid form. Pitch in keeping the lab clean; your help will be appreciated and the lab's productivity will be enhanced. If working with liquid nitrogen stocks, place cryovials in -20 freeze-racks (bottom level of freezer) while using. Return to liquid nitrogen promptly after use. Always turn off the spectrophotometer after use. Equipment, supplies and reagents should not leave our lab for use by others or for your courses; each lab and course has its own budget. The tall chair belongs at the laminar flow hood. If you move it to work in the hood, be sure to return it to its proper place when you're finished. Spray down surfaces used for microbial work. After using the analytical balance, clean up dropped media particles around the balance using brush and a tissue paper. If microwaving media, never use glassware with a top. Use a beaker much larger than the prepared volume AND. Always watch the microwaving; media with agar loves to boil out of the beaker. If so, clean up (carefully).
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VIDEO TUTORIAL http://www.youtube.com/watch?v=ORT01wl8vlY
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CHAPTER 12 BIOINFORMATICS
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Bioinformatics is a branch of biotechnology which deals with the study of methods for storing, retrieving and analyzing biological data, such as nucleic acid (DNA/RNA) and protein sequence, structure, function, pathways and genetic interactions. It generates new knowledge that is useful in such fields as drug design and development of various new software tools. Bioinformatics also deals with algorithms, databases and information systems, web technologies, artificial intelligence and soft computing, information and computation theory, structural biology, software engineering, data mining, image process.
http://www.ncbi.nlm.nih.gov/
Just surf and surf this website for as much knowledge in biotechnology. The pubmed has lot of journals. Research papers for study purposes you will find ample of information you looking for and these days bioinformatics makes our life a bit easier for us. We can use its taxonomy browser for browsing the taxonomic classification. And first thing to do on the NCBI is click on the link getting started and reading the available tutorials and the handbooks, they even have incorporated video tutorials on YouTube you can find the link on the website itself. There own search engine like Google is ENTREZ
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http://blast.ncbi.nlm.nih.gov/Blast.cgi The Basic Local Alignment Search Tool (BLAST) finds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. (Source taken from above page)
This blast is the most widely used and important tool in biotechnology.
VIDEO TUTORIAL http://www.youtube.com/watch?v=rlK-5joOlyU&feature=related
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EXAMPLE
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HOW TO START WITH ANY EXPERIMENT? Selection of topic Thorough background research about the topic. Go step wise make a template for each small experiment. Sample template: Template Aim: making media NA (Nutrient Agar) Requirements (note them down after thorough research and in detail): NA powder (or nutrient broth will be available add agar to it), spatula, weighing machine, amount of media required, 2litre glass bottle, magnetic stirrer. Foil or paper (to weigh the ingredients), autoclave tape, cap Flow chart procedure:
weighing the required amount of constituents
put it in the bottle
measure the amount of water and pour in the bottle
mix using a magnetic stirer
seal and label it autoclaving
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Use of NA: Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms in water, sewage, faeces and other materials. Composition: (available on the media powder bottle) Ingredients Gms / Litre Peptic digest of animal tissue 5.000 Beef extract 3.000 Agar 15.000 Final pH (at 25°C) 6.8±0.2 Suspend 23.00 grams in 1000 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving At 15 lbs pressure (121°C) for 15 minutes. NOTES: Additional info arising from time to time should be noted down here: References: http://himedialabs.com/TD/M561.pdf
(useful for further reference and for making final reports)
Save it ms word and also take a printout for experiment record book BE ORGANISED FOR BEST RESULTS. ALL THE BEST
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REFERENCES http://www.biotopics.co.uk/microbes/tech1.html https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=608781&tab=Overview &catID=608781&tab=Overview http://www.microbelibrary.org/component/resource/laboratory-test/3085-preparing-spread-plates-protocols http://biology.kenyon.edu/courses/biol09/tetrahymena/serialdilution3.htm http://www.sciencebuddies.org/science-fair-projects/project_ideas/MicroBio_p015.shtml http://nsdl.niscair.res.in/bitstream/123456789/133/2/Microorganism_studymethods.pdf http://science.pc.athabascau.ca/labinfo.nsf/0/1c5d8f30b6bfedd587256cd1005ca5e7?OpenDocument http://www.sumanasinc.com/webcontent/animations/content/streakplate.html http://www.appliedbiosystems.com/absite/us/en/home.html
PCR protocols: a guide to methods and applications.,Innis, M. A.;Gelfand, D. H.;Sninsky, J. J.;White, T. J. 1. Ausubel, F.M., et al. (1987)Current Protocols in Molecular Biology. New York: John Wiley and Sons. 2. Animal Genome Size Database— www.genomesize.com 3. Systma, K.K., Givnish, T.J.,Smith, J.F., and Hahn, W.J. (1993) Collection and storage of land plant samples for macromolecularcomparisons. Methods Enzymol. 234, 23. 4. Wilfinger, W.W., Mackey, M.,and Chomczynski, P. (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques22, 474. 5. Sambrook, J., Fritsch, E.F., andManiatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. 6. Critical Factors for Successful PCR, QIAGEN http://www.ncbi.nlm.nih.gov/guide/all/#howtos_ ^ Paulien Hogeweg (2011). "The roots of Bioinformatics in Theoretical Biology". The NCBI Handbook Editors: Jo McEntyre Jim Ostell National Center for Biotechnology Information
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