All section of lab methods level 3 2017 2018 by Mona Moghazee

Genetics Department

Practical Course of Laboratory Methods Level 3

(English program) 2017-2018

The identification form : The distribution of practical lessons on the topics scheduled weeks of the semester in undergraduate table. Course Name: Laboratory Methods No. decision: Semester: 2nd The Level: 3 Division: Biotechnology English Teaching Committee: Prof. Dr. Ahmed Hussein Abu Doma, Prof. Dr. Khaled Fahmy, Mona Moghazee, Hala Zoghly and Hader Yousry The goal of the study of decision:

Weeks Title 1

Laboratory equipment

Preparation of solution

Southern blotting

Northern blotting

Western blotting

Differential display

Quiz

Microarray

In situ hybridization

Sequencing technology

Revision

Final exam

Laboratory Methods, Level 3

Day

Lab. 1 Laboratory Equipment Lab. Methods level-3 Main Topics: • Priority • Sign of Priority • Priority of Laboratory Equipment Much of this equipment is already available in many biochemical and plant biology laboratories, especially given that PCR technology has become a standard tool in almost any area of biological research. However, some items have relatively specific uses and are not generally found in a nonmolecular biology laboratory. Laboratory Priorities: assigning priorities to requested laboratory tests allows the laboratory to properly triage the testing. To help the reader to set priorities when purchasing equipment for a new laboratory, the importance of each item is classified as follows, with application range as well as available alternatives given within parentheses: Sign

CLON HYB RAD

Priority Essential Highly recommended Helpful or optional Required for tissue culture and/or molecular cloning experiments Required for experiments that involve blot hybridization Required for experiments that involve the use of radioisotopes

Laboratory Methods, Level 3

Laboratory Equipment

Image

Water bath

Thermal cycler

Thermostatcontrolled metal heating blocks

Dry incubator or rotary shaker

Hybridization oven for rotating tubes

Laboratory Methods, Level 3

use

priorit y

growing plant cells, labeling probes, blot hybridization, restriction, ligation, incubation of extracts in some DNA isolation methods. for automated PCR reactions; heating of probes and samples.

hold test tubes and/or centrifuge tubes (for small-scale DNA extraction, incubation of sample or DNA restriction at on dry heat) for growing bacterial CLON, cells, HYB blot hybridization, washing Southern blots, staining agarose gels for Southern blot and HYB gel hybridizations; sealed bags in a thermo controlled rotary shaker or water bath can also be used.

Autoclave

leak-proof autoclaving bags, sterilization tape (to label sterilized solutions, glassware, or other items).

Filters and filtration units

to sterilize solutions that cannot be autoclaved, e.g., vitamins, glucose.

Oven

to sterilize glassware

Distilled Water

Deionization or water purification.

Centrifuges:

Microfuges Low-speed centrifuge (10,000 to 20,000 rpm). Used as a step in DNA extraction, PCR, and many other applications.

Refrigerator 4Ë&#x161;C

to store, e.g., solutions, DNA, PCR products

Laboratory Methods, Level 3

Freezer 20˚C

Freezer–70 to – 80˚C

to store, e.g., solutions, aliquots of PCR reagents, deoxynucleotide triphosphates [dNTPs], DNA, Taq DNA poly-merase, restriction enzymes, and other DNAmodifying enzymes for long-term storage of frozen tissue samples, reagents, DNA, bacterial cells, X-ray cassettes for autoradiography

Isothermal storage containers for liquid nitrogen

needed for many DNA isolation procedures.

Horizontal Gel Electrophoresis Equipment

For agarose gel

Vertical Gel Electrophoresis Equipment

for polyacrylamide gels (e.g., for AFLP and microsatellite analysis). Or protein electrophoresis.

Laboratory Methods, Level 3

Gel documentation system

visualize fragments

Spectrophotometer

Determination of sample quantity and quality.

(UV and visible light)

Common laboratory glassware and plasticware including beakers, graduated measuring cylinders, Erlenmeyer flasks, reagent bottles of various sizes

DNA

All laboratory tasks

PCR tubes and caps

Carrying polymerase chain reaction components

Microfuge tubes and caps (0.2, 0.5, 1.5, and 2.0 ml)

Carrying different samples for different applications.

Laboratory Methods, Level 3

Micropipettes and disposable pipet tips

Adjust the sizes used

Microwave oven

for melting agar and agarose

pH meter

To adjust pH

Thermometers

To adjust meager the degree of temperature.

Magnetic heater/stirrer

Liquid mixing

Analytical balance

To measure the mass

Laboratory Methods, Level 3

Mortars pestles

and

For grinding material

cell

Vortex mixer

Mixing and grinding

Alcohol/waterp roof markers

Labeling sample tubes

Laboratory timers and stopwatches

Management the time

Dishwashing machine

Cleaning the Dishes

CLON Bunsen burner

Laboratory Methods, Level 3

To sterilize inoculation loops

Lab. 2 Preparation of laboratory solution Lab. Methods level-3 Main Topics: • Chemicals 1. Biosafety lab code 2. MSDS = material safety data sheet 3. calculate the concentration • What’s different between: 1. Solution & Buffer. 2. Solute & Solvent. • Workout. • Exercise at home.

Chemicals 1. Biosafety lab code:

…………

MSDS = Material Safety Data Sheet

A Material Safety Data Sheet (MSDS) is a document that contains information on the potential hazards (health, fire, reactivity and environmental) and how to work safely with the chemical product. It is an essential starting point for the development of a complete health and safety program. It also contains information on the use, storage, handling, and emergency procedures all related to the hazards of the material. The MSDS contains much more information about the material than the label. MSDSs are prepared by the Laboratory Methods, Level 3

supplier or manufacturer of the material. It is intended to tell what the hazards of the product are, how to use the product safely, what to expect if the recommendations are not followed, what to do if accidents occur, how to recognize symptoms of overexposure, and what to do if such incidents occur.

What information is on the MSDS? There are nine (9) categories of information that must be present on an MSDS in Canada. These categories are specified in the Controlled Products Regulations and include: 1. Product Information: product identifier (name), manufacturer and supplier's names, addresses, and emergency phone numbers 2. Hazardous Ingredients 3. Physical Data 4. Fire or Explosion Hazard Data 5. Reactivity Data: information on the chemical instability of a product and the substances it may react with 6. Toxicological Properties: health effects 7. Preventive Measures 8. First Aid Measures 9. Preparation Information: who is responsible for preparation and date of preparation of MSDS Laboratory Methods, Level 3

The Controlled Products Regulations prescribes information must be present in more detail.

what

Calculate the concentration: Preparing Chemical Solutions: Lab experiments and types of research often require preparation of chemical solutions in their procedure.

Solution 1: Percent Solutions Formula: Mass percent solutions are defined based on the grams of solute per 100 ml of solution. Example: A 10% NaCl solution has ten grams of sodium chloride dissolved in 100 ml of solution. How to prepare NaCl â&#x20AC;&#x153;Percent Solutionsâ&#x20AC;?? â&#x20AC;˘ Procedure: 1. Weigh 10g of sodium chloride. 2. Pour it into a graduated cylinder or volumetric flask containing about 80ml of water. 3. Once the sodium chloride has dissolved completely (swirl the flasks gently if necessary), add water to bring the volume up to the final 100 ml.

Solution 2: Dilutions 1. Dilution by water: Formula: Stock solutions are often prepared and must be diluted to final concentration. An equation relating the various parameters is as follows:

Formula: C1 x V1 = C2 x V2 Laboratory Methods, Level 3

V1 = volume of stock V2 = final volume needed. reagent. C1 = concentration of the C2 = final concentration stock reagent. needed. Example Calculate the total volume of solution needed to get an 80% solution from of 95% ETOH (Ethanol) in volume 100ml? Procedure: 1- C1 x V1 = C2 x V2 V1= (80 x 100)/ 95 95 x? =80 x 100 V1 = 84.21 2- Take 84.21 ml from Ethanol add water to bring the volume up to the final 100 ml.

2. Dilution by mixing different concentrations: Cf x Vf = (C1 x V1) + (C2 x V2) Where C = concentration, V = volume, f = final solution, 1 = 1st solution and 2 = 2nd solution Example: if we add 20 ml of 1% NaCl to 30 ml of 2% NaCl the final volume will be 50 ml and the final concentration will be 1.6% Cf? x Vf = C1 x V1 + C2 x V2 Cf = [C1 x V1 + C2 x V2] / Vf Cf = [1 x 20 + 2 x 30] / 50 = 1.6%

3. Dilution By mixing different solutions: Cb x Vb = Ca x Va Where C = concentration, V = volume, b = before mixing and a = after mixing Example: if we add 20 ml of 1% KCl to 30 ml of 2% NaCl the final volume will be 50 ml and the final concentration of NaCl will be 1.2% and for KCl will be 0.4% Cb x Vb = Ca x Va Ca = Cb x Vb / Va Laboratory Methods, Level 3

Ca [for NaCl] = 2 x 30 / 50 = 1.2% Ca [for KCl] = 1 x 20 / 50 = 0.4%

Solution 3: Molar Solutions Molar solutions are the most useful in chemical reaction calculations because they directly relate the moles of solute to the volume of solution. Formula: g = M x MW x L Avoid the following: 1. Caution: Do not simply measure 100ml of water and add 10g of sodium chloride. This will introduce error because adding the solid will change the final volume of the solution and throw off the final percentage. 2. Caution: you should be sure dissolve the salt in solvent

Laboratory Methods, Level 3

Workout How to prepare 50mM Tris buffer? G= MW X M X L G= 121.14 X (50/1000) X (100/1000)=

6.057 g How to prepare 5M EDTA buffer? G= MW X M X L G = 292.24 X (5) X (20/1000) = 29.224

Some exercise How to make 100 mM Tris solutions in 80ml of water. (MW: 121.14) …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… How to make 50% of NaCl in volume 100ml. …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… Make 15% of isopropanol from conc. 75% in volume 50ml. …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… ……………………………………………………………………………

Laboratory Methods, Level 3

Homework 1. How much ethanol is present in 200 mL of a 25% (v/v) solution of ethanol? ……………………………………………………………………… ……………………………………………………………………… ……………………………………………………………………… ……………………………………………………………………… 2. How can prepare this reagents and buffers:

…………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… Some Resources: • •

http://www.ccohs.ca/oshanswers/legisl/msdss.html http://voyager.dvc.edu/~lborowski/chem108index/Solutions/Solution_L ecture_2.htm

Laboratory Methods, Level 3

Practical work sheet No.1

Laboratory Methods, Level 3

Lab.3 Blotting technology Southern blotting tchenique Lab. Methods level-3 Main point: Tool Researcher Techquie Device

Tool: "Syringe" A Syringe "dead volume", we will be performing a gene therapy mouse experiment for which we will be injecting mice with our viral construct. Unfortunately, the syringe we are using has a dead volume of about 60ul which adds up to quite a lot of waste. As a result, I'm looking for a new syringe or syringe tip. Explain technique that works on this tool? ……………………………………… ……………………………………… ……………………………………… ………………………………………

Technology or Technique or Terms blotting "

"Southern

Southern blotting Definition:

Laboratory Methods, Level 3

The southern blotting principle is Hybridization, which is the process of forming a double stranded DNA molecule between a single-stranded DNA probe and a single-stranded target DNA. Southern blotting is applied for several aspects of molecular biology. It is useful in RFLP mapping, forensic studies, DNA methylation in gene expression, detection of mutated genes in genetic disorders, DNA fingerprinting, etc. Southern blotting steps: 1. 2. 3. 4. 5. 6. 7. 8.

Extract and purify DNA from cells. DNA is restricted with enzymes. Separated by electrophoresis. Denature DNA. Transfer to nitrocellulose paper. Add labeled probe for hybridization to take place. Wash off unbound probe. Autoradiograph.

The major improvement has been the introduction of nylon membranes, which have three advantages over their nitrocellulose counterparts: 1. Nylon membranes are less fragile than nitrocellulose sheets, and Nylon membranes cannot be damaged by handling and a single blot can be rehybridized up to ten times, this limit being due not to eventual breakage of the membrane but to the gradual loss of the blotted DNA during repeated hybridizations. 2. Nylon membranes is that under certain conditions (a positively charged membrane and an alkaline transfer buffer) the transferred DNA becomes covalently bound to the membrane during the transfer process 3. Nylon membranes efficiently bind DNA fragments down to 50 bp in length, whereas nitrocellulose membranes are effective only with molecules longer than 500 bp. Laboratory Methods, Level 3

Southern blotting advantage • Effective way to detect a specific DNA sequence in large, complex sample of DNA. • Can be used to quantify the amount of the present DNA. • Cheaper than DNA sequencing.

Southern blotting dis-advantage • More expensive than most other tests. • Complex and labor intensive.

Southern blotting applications: 1. To identify specific DNA in a DNA sample. 2. To Isolate desired DNA for construction of rDNA. 3. Identify mutations, deletions, and gene rearrangements. 4. In DNA fingerprinting

Researcher "Susumu Tonegawa" Susumu Tonegawa is a Japanese molecular biologist who was awarded the Nobel Prize for Physiology or Medicine in 1987. Renowned for his discovery of the genetic mechanism that produces antibody diversity, his work elucidated the genetic mechanism of the adaptive immune system. Laboratory Methods, Level 3

Device "Spectrophotometry" Spectrophotometer is the quantitative measurement of how much a chemical substance absorbs light by passing a beam of light through the sample using a spectrophotometer. Concepts A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector.

Applications in biochemistry Spectrophotometry is an important technique used in many biochemical experiments that involve DNA, RNA, and protein isolation, enzyme kinetics and biochemical analyses. A brief explanation of the procedure of spectrophotometry includes comparing the absorbency of a blank sample that does not contain a colored compound to a sample that contains a colored compound. Explain the technique that works on this device? …………………………………………………………………………… …………………………………………………………………………… Laboratory Methods, Level 3

…………………………………………………………………………… …………………………………………………………………………… What are some potential sources of error in absorbance measurements? • Spectrophotometric measurements are affected by many factors, such as the type of solvent used, temperature, wavelength of light at which the measurements are made, and presence of impurities in the sample being studied. To eliminate absorbance due to the solvent, the spectrophotometer is always zeroed (tared) against the solvent. • Light scattering is due to suspended particles. The particulates will deflect light rays and cause an artificial increase in absorbance. • To avoid light scattering, it is frequent practice to centrifuge the sample before measuring absorbance to remove particulates. References: http://www.differencebetween.com/difference-between-southern-and-vsnorthern-and-vs-western-blotting/ https://www.biology-online.org/ http://w3.marietta.edu/~spilatrs/biol309/labexercises/Spectrophotometry. pdf

Laboratory Methods, Level 3

Lab.4 Blotting technology Northern blotting technique Lab. Methods level-3 Main point: Tool Researcher Techquie Device

Tool: "Needle of inculcation" It is a laboratory equipment used in the field of microbiology to transfer and inoculate living microorganisms. Explain technique that works on this tool? ……………………………………… ……………………………………… ……………………………………… ……………………………………… ……………………………………… ……………………………………… ……………………………………… ………………………………………

Technology or Technique or Terms "Northern blotting " Northern blotting Definition: The Northern blotting principle is Hybridization, which is the process of forming a specific RNA molecule between a single-stranded RNA probe and a single-stranded target DNA. Northern blotting is a method designed to detect a specific RNA sequence or mRNA sequence from a sample to study Laboratory Methods, Level 3

gene expression. This technique was developed by Alwine, Kemp, and Stark in 1979. It differs from the southern and western blotting techniques due to several steps. However, this technique is also performed via gel electrophoresis, blotting, and hybridization with specific labeled probes and detection. Northern blotting steps: 1. Extract and purify mRNA from cells. 2. Separated by electrophoresis. 3. this gel is immersed in depurination buffer for 5-10 minute then washed with water. 4. Transfer to amino-benzyloxymethyl filter paper. 5. After transfer, the membrane is baked at 80áľ&#x2019;c 6. Add labeled probe for hybridization to take place. 7. Wash off unbound probe. 8. Autoradiograph.

Laboratory Methods, Level 3

Northern blotting applications: Northern blotting is a useful tool in detection and quantification of hybridized mRNA, studying RNA degradation, evaluation of RNA half-life, detection of RNA splicing, studying gene expression, etc. 1. To identify specific mRNA in sample. 2. To screen recombinants by detecting the mRNA produced by the transgene 3. Diseases diagnosis 4. Gene expression studies

Researcher "Norman Borlaug" One of the most prominent biologists of the 20th century, Norman Borlaug is often referred to as “The Man Who Saved a Billion Lives” and why wouldn’t he be, After all he is the man behind the Green Revolution which revolutionized the way farming was being conducted all over the world. Having spent his childhood in a large farm raising cattle and growing food crops, the young Norman had a great thirst for knowledge. Though as a kid he attended a small school, he decided to pursue his higher education from the University of Minnesota. After completing his studies, he could have opted for a lucrative job in the chemical industry, but he chooses to go to Mexico to research on creating high yielding crops. Over the course of his work he successfully developed disease resistant, high yielding wheat varieties which when combined with modern agricultural production techniques could dramatically change the way farming was done. His discoveries and production techniques helped several Laboratory Methods, Level 3

hitherto food-deficient countries in Asia and Africa to achieve food security and helped to provide food for millions of starving people. He was presented with the Nobel Peace Prize for his immeasurable contributions towards feeding millions of people around the world.

Device "Numerical pH meter" A pH meter is a scientific instrument that measures the hydrogen-ion activity in waterbased solutions, indicating its acidity or alkalinity expressed as pH. The pH meter measures the difference in electrical potential between a pH electrode and a reference electrode, and so the pH meter is sometimes referred to as a "potentiometric pH meter". The difference in electrical potential relates to the acidity or pH of the solution. The pH meter is used in many applications ranging from laboratory experimentation to quality control. The exact meaning of the "p" in "pH" is disputed, but according to the Carlsberg Foundation, pH stands for "power of hydrogen".

Applications The rate and outcome of chemical reactions taking place in water often depends on the acidity of the water, and it is therefore useful to know the acidity of the water, typically measured by means of a pH meter. Knowledge of pH is useful or critical in many situations, including chemical laboratory analyses. pH meters are used for soil measurements in agriculture, water quality for municipal water supplies, swimming pools, environmental remediation; brewing of wine or beer; manufacturing, healthcare and clinical applications such as blood chemistry; and many other applications. Laboratory Methods, Level 3

Advantage of Numerical pH meter: Fast, Accurate, and Auto Calibrated instrument used for adjustment of pH. Explain the technique that works on this device? ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… ………………………………… …………………………………

References: http://www.differencebetween.com/difference-between-southern-and-vsnorthern-and-vs-western-blotting/ https://www.biology-online.org/ http://frasesreflexionespensamientosange.blogspot.com.eg/2015/11/hermo sas-iniciales.html https://www.thinglink.com/scene/655091122904760322

Laboratory Methods, Level 3

Lab.5 Blotting technology Western blotting technique Lab. Methods level-3 Main point: Tool Researcher Techquie Device

Tool: "Wire gauze" It is a sheet of thin metal. wire gauze sits on the iron ring to provide a place to stand a beaker. on older wire gauze, the white material was asbestos currently it is a ceramic.

Explain technique that works on this tool? ………………………………………………………………………………………… ………………………………………………………………………………………… …………………………………………………………………………………………

Technology or Technique or Terms "Western blotting " Western blotting Definition: Western blotting is a method of detecting a specific protein from a protein mixture by the use of labeled antibody. Therefore, western blot is also known as an immunoblot. This technique was introduced by Towbin et al in 1979 and

Laboratory Methods, Level 3

it is now routinely performed in the labs for protein analysis. Steps are as follows. Western blotting can produce qualitative and semiquantitative data about the protein of interest. It is an important technique used in cell and molecular biology. It enables the researchers to identify the specific protein from mixture of proteins extracted from cells as well as evaluation of their size and amount. The SDS PAGE technique is prerequisite for western blotting.

Western blotting steps Tissue preparation Gel electrophoresis Transfer Blocking: steps must be taken to prevent interactions between the membrane and the antibody used for detection of the target protein (since the antibody is a protein itself). Blocking of non-specific binding is achieved by placing the membrane in a dilute solution of protein ▪ Detection (two steps, one step) ▪ Analysis: 1. Colorimetric detection: This method depends on incubation of the Western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a distinct color that precipitates next to the enzyme and thereby stains the membrane. Protein levels are evaluated through densitometry or spectrophotometry. 2. Chemiluminescent detection: This method depends on incubation of the Western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD ▪ ▪ ▪ ▪

Laboratory Methods, Level 3

cameras. Densitometry analyzes the image. Newer software allows further data analysis such as molecular weight analysis if appropriate standards are used. 3. Radioactive detection: Radioactive labels do not require enzyme substrates, but rather allow the placement of medical X-ray film directly against the western blot which develops as it is exposed to the label and creates dark regions which correspond to the protein bands of interest very expensive, health and safety risks are high 4. Fluorescent detection: The fluorescently labeled probe is excited by light and the emission of the excitation is then detected by a photosensor such as CCD camera. Allows further data analysis such as molecular weight analysis and a quantitative western blot analysis. Western blotting steps: 1. Proteins are extracted from the sample 2. Proteins are separated by their sizes using polyacrylamide gel electrophoresis 3. Separated molecules are transferred into a Polyvinylidene Difluoride "PVDF" membrane or nitrocellulose membrane by electroporation 4. The membrane is blocked for nonspecific binding with the antibodies 5. Transferred proteins are bound with primary antibody (enzyme labeled antibodies). 6. The membrane is washed to remove nonspecifically bound primary antibodies 7. Bound antibodies are detected by adding a substrate and detecting the colored precipitate formed. **Different between Nitrocellulose membranes and PVDF membranes: Nitrocellulose membranes are cheaper than Laboratory Methods, Level 3

PVDF but are far more fragile and do not stand up well to repeated probing. 1. One major difference between nitrocellulose and PVDF membranes related to the ability of each to support "stripping" antibodies off and reusing the membrane for subsequent antibody probes. 2. PVDF is sturdier and can be more reused. 3. Another difference is that, unlike nitrocellulose, PVDF must be soaked in 95% ethanol, isopropanol or methanol before use.

Advantages â&#x2013;Ş Sensitivity Because of its ability to detect as little as 0.1 Nano grams of protein in a sample, the technique can theoretically serve as an effective early diagnostic tool, sensing even the slightest immunogenic response from a virus or bacteria in a patient sample. â&#x2013;Ş Specificity

Laboratory Methods, Level 3

Disadvantages Excessive cost and technical demand

Western blotting Uses ➢ It is most sensitive and specific test for determining size and amount of protein present in any material. ➢ The confirmatory HIV test employs a western blot to detect anti-HIV antibody in a human serum sample. ➢ A western blot is also used as the definitive test for Creutzfeldt-Jakob Disease, Lyme disease, Hepatitis B infection and HSV-2 (Herpes Type 2) infection.

Researcher "William Harvey" William Harvey was a 17th-century British physician who became the first to document an understanding of blood circulation. William Harvey, physician to two consecutive kings, studied blood circulation, and his Anatomical Exercise on the Motion of the Heart and Blood in Animals (1628) recorded his findings. Though Harvey understood that the heart pumped blood into the circulatory system, he had no knowledge of the influence of oxygen in the blood nor knowledge of the existence of capillaries. It took a further two centuries before a mammalian egg was finally observed, but nonetheless Harvey's theory won credibility during his lifetime.

Laboratory Methods, Level 3

Device "Water bath" A water bath is a device that maintains water at a constant temperature. It is used in the microbiological laboratory for incubations.

Precautions 1. It is not recommended to use water bath with moisture sensitive or pyrophoric reactions. Do not heat a bath fluid above its flash point. 2. Water level should be regularly monitored and filled with distilled water only. This is required to prevent salts from depositing on the heater. 3. Disinfectants can be added to prevent growth of organisms. 4. Raise the temperature to 90 Â°C or higher to once a week for half an hour for decontamination. 5. Markers tend to come off easily in water baths. Use water resistant ones. 6. If application involves liquids that give off fumes, it is recommended to operate water bath in fume hood or in a well-ventilated area. 7. The cover is closed to prevent evaporation and to help reaching hot temperatures. 8. Set up on a steady surface away from flammable materials

Types of water bath: 1. Circulating Water Baths Circulating the water baths (also called stirrers) are ideal for applications when temperature uniformity and consistency are critical, such as enzymatic and serologic experiments. Water is thoroughly circulated throughout the bath resulting in a more uniform temperature. Laboratory Methods, Level 3

2. Non-Circulating Water Baths This type of water bath relies primarily on convection instead of water being uniformly heated. Therefore, it is less accurate in terms of temperature control. In addition, there are addons that provide stirring to non-circulating water baths to create more uniform heat transfer 3. Shaking Water Baths This type of water bath has extra control for shaking, which moves liquids around. This shaking feature can be turned on or off. In microbiological practices, constant shaking allows liquid-grown cell cultures grown to constantly mix with the air. Explain the technique that works on this device? ……………………………………… ……………………………………… ……………………………………… ……………………………………… ……………………………………… ……………………………………… ……………………………………… References: http://www.differencebetween.com/difference-between-southern-and-vsnorthern-and-vs-western-blotting/ https://www.biology-online.org/

Laboratory Methods, Level 3

Lab. 6 Gene expression methods 1.Differential display techique "DD-PCR" Lab. Methods Level-3 Main point: Tool Researcher Techquie Device

Tool: "Desiccators" The lower compartment of the desiccator contains lumps of silica gel, freshly calcined quicklime, Drierite or (not as effective) anhydrous calcium chloride to absorb water vapor. The substance needing desiccation is put in the upper compartment, usually on a glazed, perforated ceramic plate. The ground-glass rim of the desiccator lid must be greased with a thin layer of vacuum grease, petroleum jelly or other lubricant to ensure an airtight seal. • Desiccators create and maintain dry environments to ensure sample stability. • Using absorbing beads, and other desiccants, desiccator cabinets or apparatuses quickly absorb moisture, guaranteeing sensitive materials do not react to surroundings prior to testing. • In laboratory use, the most common desiccators are circular and made of heavy glass. There is usually a removable platform on which the items to be stored are placed.

Laboratory Methods, Level 3

• The desiccant, usually an otherwise-inert solid such as silica gel, fills the space under the platform. Colour changing silica may be used to indicate when it should be refreshed. Indication gels typically change from blue to pink as they absorb moisture but other colors may be used. Explain technique that works on this tool? …………………………………………………………………………… ……………………………………………………………………………

Technology or Technique or Terms "Differential display" Differential display Definition: Differential display is a powerful technique for detecting and quantitating changes in gene expression patterns between differently treated cells. Fragments of those genes which are induced or suppressed can be identified and isolated for further analysis, with no prior knowledge of the sequences involved. The technique is PCR based, and yields results in only 1-3 days. Differential display polymerase chain reaction (DDPCR) uses PCR to amplify and display cDNAs derived from the mRNAs of a given cell or tissue type. First-strandsynthesis is primed with an anchored primer complementary to ∼13 nucleotides (nt) of the poly(A) tail of mRNA and the adjacent 2 nt of the transcribed sequence. Anchored primers therefore anneal to the junction between the poly(A) tail and the 3′-untranslated region of mRNA templates. A second primer, an arbitrary sequence of ∼10nt, is then added to the reaction mixture, and doublestranded cDNAs are produced by PCR carried out at low stringency. By comparing the banding patterns of the resulting cDNA products, it is sometimes possible to identify the products of differentially expressed genes.

Laboratory Methods, Level 3

In this approach: the 3′ termini of eukaryotic messenger RNA are systematically amplified by reverse transcriptionPCR using one of the three anchored oligo-dT primers (in this example, 5′-AAGCTTTTTTTTTTTG -3′) in combination with a set of short primers of arbitrary sequences (in this example, 5′-AAGCTTGATTGCC-3′). The length of the arbitrary primers is designed such that each will recognize about 50–100 mRNAs under a given PCR condition. As a result, mRNA 3′ tails, defined by any given pair of anchored primer and arbitrary primer, are amplified. By changing primer sequences, different subsets of mRNA can be analyzed and displayed by denaturing polyacrylamide gel electrophoresis. Side-by-side comparisons of such complementary DNA patterns between or among relevant RNA samples indicate differences in gene expression. Differentially expressed cDNA bands can be retrieved and sequenced for further molecular characterization.

Laboratory Methods, Level 3

Differential display advantage DD-PCR offers several advantages, including the rapidity and sensitivity of the assay. Only small quantities of RNA are needed to compare several conditions or variables simultaneously, and to identify differentially expressed genes in more than one population.

Differential display dis-advantage irreproducibility and false positive readings may result under the low PCR stringency conditions used for nonspecific primers

Differential display applications: 1. Identification of transcripts that are differentially regulated. 2. Essential step prior to carry out whole transcriptome sequencing 3. Validation of RNA transcripts isolated from a whole transcriptome library

Researcher "David Baltimore" • Famous As: Virologist • He is an American biologist who won a share of the 1975 Nobel Prize in Physiology or Medicine. As a researcher, he has made tremendous contributions to immunology, virology, cancer research, biotechnology, and recombinant DNA research. • he performed pioneering research on animal virology. Later in his career, he independently discovered reverse transcriptase, an enzyme that synthesizes DNA from RNA.

Laboratory Methods, Level 3

• And researched on interaction between viruses and the genetic material of the cell.

Device "Distilled water machine " Equipment used for water purification and distillation includes deionized (DI) water systems, water distillers, reagent-grade water systems, and laboratory filters. DI and distilled water are the most common types of purified water used in the lab, but techniques also used to produce highpurity water include: • Carbon filtration • Ion exchange • Reverse osmosis (RO) • Ultrafiltration • UV oxidation Explain the technique that works on this device? …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… References: http://www.mdpi.com/2079-7737/5/2/20/htm https://www.nature.com/articles/nrc1214/figures/3 https://www.labcompare.com/General-Laboratory-Equipment/942-WaterDistillation-Equipment-Water-Purification-Equipment/ https://www.nationaldiagnostics.com/electrophoresis/article/differentialdisplay

Laboratory Methods, Level 3

Lab. 7 Gene expression methods 2.Microarray techique "cDNA microarray " Lab. Methods Level-3 Main point: Tool Researcher Techquie Device

Tool: "Magnetic Stir Bars" A stir bar is the magnetic bar placed within the liquid which provides the stirring action. The stir bar's motion is driven by another rotating magnet or assembly of electromagnets in the stirrer device, beneath the vessel containing the liquid. Stir bars are typically coated in Teflon, or less often in glass. Glass coatings are used for liquid alkali metals (except lye, which will eat through glass) and alkali metal solutions in ammonia. Both coatings are chemically inert and do not contaminate or react with the reaction mixture they are in Explain technique that works on this tool? …………………………………………………………………………… ……………………………………………………………………………

Technology or Technique or Terms "Microarrays, DNA Microarrays, cDNA Microarrays" Microarrays Definition: The technique of microarrays came into use in the late 1990s. Many cDNA clones were available from selected model organisms. Many cDNAs can be spotted onto an array that is Laboratory Methods, Level 3

used to determine changes in the level of expression of all cDNAs in a comparison of two samples.

A DNA microarray consists of an orderly arrangement of DNA fragments representing the genes of an organism. Each DNA fragment representing a gene is assigned a specific location on the array, usually a glass slide, and then microscopically spotted (less than 1 mm) to that location. Using highly accurate robotic spotters, over 30,000 spots can be placed on one slide. • DNA microarrays can be used to detect DNA (as in comparative genomic hybridization), or detect RNA (most commonly as cDNA after reverse transcription) that may or may not be translated into proteins. • The process of measuring gene expression via cDNA is called expression analysis or expression profiling. • The traditional solid-phase array is a collection of orderly microscopic "spots", called features, each with thousands of identical and specific probes attached to a solid surface, such as glass, plastic or silicon biochip (commonly known as a genome chip, DNA chip or gene array). Thousands of these features can be placed in known locations on a single DNA microarray. • The alternative bead array is a collection of microscopic polystyrene beads, each with a specific probe and a ratio of two or more dyes, which do not interfere with the fluorescent dyes used on the target sequence.

Laboratory Methods, Level 3

The experiment to measure high temperature

we compare the mRNAs of control cells to mRNAs from cells exposed to high temperature. As shown below, the mRNAs from the two samples are made into cDNA.

1. RNA extraction cDNA synthesis.

and

3. Complementary DNA

Laboratory Methods, Level 3

2. Make cDNA probes by Cys3 and Cys5

4. Photograph the microarray using a microscope while illuminating the array with UV light.

1. 2. 3. 4. 5.

Microarray advantage Provides data for thousands of genes One experiment instead of many Fast and easy to obtain results Huge step closer to discovering cures for diseases and cancer Different parts of DNA can be used to study gene expression

1. 2. 3. 4. 5.

Microarray dis-advantage Correlations in results do not mean causation Very little knowledge is available about many genes Just because mRNA is "turned on" doesn't mean proteins are made It will the findings lead to unethical medical procedures. Scientists have no standardized way to share results.

Microarray applications: 1. Gene expression profiling: In an mRNA or gene expression profiling experiment the expression levels of thousands of genes are simultaneously monitored to study the effects of certain treatments, diseases, and developmental stages on gene expression. 2. Chromatin immunoprecipitation "CHIP" on Chip: DNA sequences bound to a particular protein can be isolated by immunoprecipitating that protein (ChIP), these fragments can be then hybridized to a microarray (such as a tiling array) allowing the determination of protein binding site occupancy throughout the genome. 3. SNP detection: Identifying single nucleotide polymorphism among alleles within or between populations. Several applications of microarrays make use of SNP detection, including genotyping, forensic analysis, measuring predisposition to disease, identifying drug-candidates, evaluating germline mutations in individuals or somatic mutations in cancers, assessing loss of heterozygosity, or genetic linkage analysis. Laboratory Methods, Level 3

Researcher "Marguerite Vogt" • Famous As: American cancer biologist and Virologist at the ‘Salk Institute for Biological Studies’. • She collaborated with Nobel Prizewinning scientist Renato Dulbecco to analyze the way polio virus develops plagues in cell cultures, a discovery that eventually aided in the development of a polio vaccine. • The duo examined how some viruses take control of the cells infected by them. They displayed that polyomavirus, the small DNA viruses that are characteristically extensive, tucks in their own DNA into that of the host cell. These analyses of the duo changed the descriptive form of virology to a more determinable one. • Vogt and Dulbecco also showed the way a virus can change a cell to a cancerous one.

Device "Magnetic stirrer" • A magnetic stirrer or magnetic mixer is a laboratory device that employs a rotating magnetic field to cause a stir bar (also called "flea") immersed in a liquid to spin very quickly, thus stirring it. • Magnetic stirrers are often used in chemistry and biology.

Laboratory Methods, Level 3

Explain the technique that works on this device? …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… References: https://www.thefamouspeople.com/profiles/marguerite-vogt-7505.php Adomas A; Heller G; Olson A; Osborne J; Karlsson M; Nahalkova J; Van Zyl L; Sederoff R; Stenlid J; Finlay R; Asiegbu FO (2008). "Comparative analysis of transcript abundance in Pinus sylvestris after challenge with a saprotrophic, pathogenic or mutualistic fungus". Tree Physiol. 28 (6): 885– 897. doi:10.1093/treephys/28.6.885. PMID 18381269. Hacia JG; Fan JB; Ryder O; Jin L; Edgemon K; Ghandour G; Mayer RA; Sun B; Hsie L; Robbins CM; Brody LC; Wang D; Lander ES; Lipshutz R; Fodor SP; Collins FS (1999). "Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays". Nat Genet. 22 (2): 164–167. doi:10.1038/9674. PMID 10369258.

Laboratory Methods, Level 3

Lab. 8 Gene expression methods 3.in situ hybridization techique Lab. Methods Level-3 Main point: Tool Researcher Techquie Device

Tool: "iris scissors" • Iris scissors are a type of scissors with short blades that was originally developed for ophthalmic surgery. • Iris scissors are also available in the crafting market and are sometimes used for the production of fabricrelated goods. Explain technique that works on this tool? …………………………………………………………………………… ……………………………………………………………………………

Technology or Technique or Terms hybridization" in situ hybridization Definition:

"in situ

In molecular and cell biology, in situ It means "locally", "on site", "on the premises" or "in place" to describe an event where it takes place and is used in many different contexts. For example, in fields such as biology, in situ may describe the way a measurement is taken, that is, in the same place the phenomenon is occurring without isolating it from other systems or altering the original conditions of the test. • In situ hybridization (ISH): is a type of hybridization that uses a labeled complementary Laboratory Methods, Level 3

DNA, RNA or modified nucleic acids strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ), or, if the tissue is small enough (e.g., plant seeds, Drosophila embryos), in the entire tissue (whole mount ISH), in cells, and in circulating tumor cells (CTCs). • It obtains temporal and spatial information about gene expression and genetic loci.

Scientific Basis of ISH 1. Double-stranded DNA denatures on heating to single-stranded DNA. On cooling, the singlestranded DNA reanneals with its complementary sequence into doublestranded DNA. 2. labeled fragment of a DNA sequence (a DNA probe) is denatured and added to denatured nuclei or chromosomes on a routine, air-dried interphase preparation during the process of reannealing, some of the labeled DNA will hybridize to its complementary sequence in the chromosomal DNA.

Types of In Situ Hybridization: While the basic workflow of ISH is like that of blot hybridizations. the nucleic acid probe is synthesized, labeled, purified, and annealed with the specific target—the difference is the greater amount of information gained by visualizing the results within the tissue. • There are two basic ways to visualize your RNA and DNA targets in situ fluorescence (FISH) and chromogenic (CISH) detection. Laboratory Methods, Level 3

â&#x20AC;˘ And there are two another way; in situ PCR and Catalyzed reporter deposition (CARD).

1. FISH technique (Direct & indirect)

(a) The basic elements of FISH are a DNA probe and a target sequence. (b) Before hybridization, the DNA probe is labeled by various means, such as nick translation, random primed labeling, and PCR. Two labeling strategies are commonly used: indirect labeling (left panel) and direct labeling (right panel). For indirect labeling, probes are labeled with modified nucleotides that contain a hapten, whereas direct labeling uses nucleotides that have been directly modified to contain a fluorophore. (c) The labeled probe and the target DNA are denatured. (d) Combining the denatured probe and target allows the annealing of complementary Laboratory Methods, Level 3

DNA sequences. (e) If the probe has been labeled indirectly, an extra step is required for visualization of the no fluorescent hapten that uses an enzymatic or immunological detection system. Whereas FISH is faster with directly labeled probes, indirect labeling offers the advantage of signal amplification by using several layers of antibodies, and it might therefore produce a signal that is brighter compared with background levels. 2. Chromogenic in situ hybridization (CISH) (indirect) it is a cytogenetic technique that combines the chromogenic signal detection method of immunohistochemistry (IHC) techniques with in situ hybridization

CISH is like FISH in that they are both in situ hybridization techniques used to detect the presence or absence of specific regions of DNA. However, CISH is much more practical in diagnostic laboratories because it uses bright-field microscopes rather than the more expensive and complicated fluorescence microscopes used in FISH. CISH probes are labelled with biotin or digoxigenin and can be detected using a bright-field microscope after other treatment steps have been applied

Technique CISH

Instrument/ visualization method Bright-field microscopy

Laboratory Methods, Level 3

Primary advantage

Primary application

Ability to view the CISH signal and tissue morphology simultaneously

Molecular pathology diagnostics

FISH

DNAFISH

Fluorescence microscopy

Multiplexable: visualize multiple targets in the same sample

Gene presence, copy number, and location; mutation analysis

RNAFISH

Fluorescence microscopy, HCS, and flow cytometry

Multiplexable: visualize multiple targets in the same sample

Gene expression, RNA temporal and spatial localization

3. in situ amplification "In situ PCR” • In situ PCR has mainly been used to identify DNA sequences that are not easy to detect using standard in situ hybridization. These sequences include human single‐copy genes, chromosomal translocations, and rearranged cellular genes. • In situ PCR is also used for mapping genomic sequences that have a low copy number in metaphase chromosomes. However, in situ PCR has multiple problems, including low efficiency of amplification and poor reproducibility. 4. Catalyzed reporter deposition (CARD) • This method involves deposition of activated biotinylated tyramine onto electron rich moieties (e.g., tyrosine and phenylalanine) at or close to the site of horseradish peroxidase (HRP). • When using HRP-labeled probes fluorescent staining results from a secondary incubation with fluorescently labeled tyramine. • The specifically bound peroxidase molecules catalyze the deposition of these labeled reporter compounds within cells targeted by the HRP-tagged probe.

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In Situ Hybridization steps:

1. Preparation of Tissue: • Treatment with proteases (proteinase K is the most common) • Acetylation of sections • Optimization of tissue processing, including fixation and storage 2. In situ hybridization probes: • Double-stranded DNA (dsDNA) probes • Single-stranded DNA (ssDNA) probes • RNA probes (riboprobes) In situ hybridization Labeling technique: • Radioactive probe 32P, 125I, 35S, or 3H • Non-radioactive labels • biotin • digoxigenin • fluorescent dye (FISH)

Laboratory Methods, Level 3

In situ hybridization In situ hybridization advantage dis-advantage • Maximum use of tissue • Difficulty in identifying that is difficult to obtain targets that have low DNA (e.g., embryos and clinical and RNA copies. However, biopsies). approaches are • Hundreds of different continually being hybridizations can be developed to improve the performed on the same sensitivity of in tissue. situ hybridization • Libraries of tissues can be formed and stored in the freezer for future use. Applications of In Situ Hybridization: 1. Microbiology 2. Pathology 3. Developmental biology 4. Karyotyping and phylogenetic analysis 5. Physical mapping

Researcher "Isaac Asimov" • Famous As: Writer, Professor • Isaac Asimov is best known as the most successful writer of science fiction and popular science books. • Asimov opened the doors for the new age of science fiction writing which the world had never tasted before him. Asimov is credited with having edited over 500 books. • Asimov was a brilliant professor of biochemistry at Boston University. Besides being a prolific writer, Asimov was also an integral part of (President) the American Humanist Association. Asimov is also known for his work as a civilian at the Philadelphia Navy Yard's Naval Air Experimental Station during the World War II. “Robotics”

Laboratory Methods, Level 3

was a term coined by Asimov which went on to become a branch of technology.

Device "Incubator" 1. In biology, an incubator is a device used to grow and maintain microbiological cultures or cell cultures. 2. The incubator maintains optimal temperature, humidity and other conditions such as the carbon dioxide (CO2) and oxygen content of the atmosphere inside. 3. Incubators are essential for a lot of experimental work in cell biology, microbiology and molecular biology and are used to culture both bacterial as well as eukaryotic cells. Explain the technique that works on this device? …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… References: https://www.nature.com/scitable/topicpage/fluorescence-in-situ-hybridization-fish-327#

Laboratory Methods, Level 3

Lab. 9 Sequencing technology Lab. Methods Level-3 Main point: Tool Researcher Techquie Device

Tool: "Spatulas-lab" Laboratory double spatulas in stainless steel to take a solid (e.g. powder). Two flat spatulas and a spoon spatula. Explain technique that works on this tool? …………………………………………………………………………… …………………………………………………………………………… …………………………………………………………………………… ……………………………………………………………………………

Technology or Technique or Terms " Sequencing technology" Sequencing technology Definition: DNA sequencing is the process of determining the precise order of nucleotides within a DNA molecule. It includes any method or technology that is used to determine the order of the four bases—adenine, guanine, cytosine, and thymine—in a strand of DNA. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. 1. Maxam-Gilbert sequencing method: Allan Maxam and Walter Gilbert published a DNA sequencing method in 1977 based on chemical modification of DNA and subsequent cleavage at specific bases. Also known as Laboratory Methods, Level 3

chemical sequencing, this method allowed purified samples of double-stranded DNA to be used without further cloning. This method's use of radioactive labeling and its technical complexity discouraged extensive use after refinements in the Sanger methods had been made. Maxam-Gilbert sequencing requires radioactive labeling at one 5' end of the DNA and purification of the DNA fragment to be sequenced. Chemical treatment then generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus, a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule. The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each corresponding to a radiolabeled DNA fragment, from which the sequence may be inferred.

(a) Preparation of tube

Laboratory Methods, Level 3

(b) Electrophoresis & reading

2. Sanger sequencing method "Chain-termination methods" Frederick Sanger is among the rare breed of individuals, who won the Nobel Prize twice - once in 1958 for identifying amino acid sequences in proteins and the second time in 1980 for DNA sequencing. The chain-termination method developed by Frederick Sanger and coworkers in 1977 soon became the method of choice, owing to its relative ease and reliability. When invented, the chain-terminator method used fewer toxic chemicals and lower amounts of radioactivity than the Maxam and Gilbert method. Because of its comparative ease, the Sanger method was soon automated and was the method used in the first generation of DNA sequencers.

Sanger sequencing is the method which prevailed from the 1980s until the mid-2000s. Over that period, great advances were made in the technique, such as fluorescent labelling, capillary electrophoresis, and general automation. These developments allowed much more efficient sequencing, leading to lower costs. The Sanger method, in mass production form, is the technology which produced the first human genome in 2001, ushering in the age of genomics. Laboratory Methods, Level 3

However, later in the decade, radically different approaches reached the market, bringing the cost per genome down from $100 million in 2001 to $10,000 in 2011.

Sequencing technology applications: 1. Molecular biology: Sequencing is used to study genomes and the proteins they encode. Information obtained using sequencing allows researchers to identify changes in genes, associations with diseases and phenotypes, and identify potential drug targets. 2. Evolutionary biology: Since DNA is an informative macromolecule in terms of transmission from one generation to another, DNA sequencing is used in evolutionary biology to study how different organisms are related and how they evolved. 3. Metagenomics: The field of metagenomics involves identification of organisms present in a body, water, sewage, dirt, debris filtered from the air, or swab samples from organisms. Knowing which organisms are present in a particular environment is critical to research in ecology, epidemiology, microbiology, and other fields. Sequencing enables researchers to determine which types of microbes may be present in a microbiome, for example. 4. Medicine: Medical technicians may sequence genes (or, theoretically, full genomes) from patients to determine if there is risk of genetic diseases. This is a form of genetic testing, though some genetic tests may not involve DNA sequencing.

Researcher "Salvador Edward Luria" Laboratory Methods, Level 3

Salvador E. Luria was an Italian microbiologist who jointly won the Nobel Prize in Physiology or Medicine in 1969 with Max DelbrĂźck and Alfred Hershey, for their discoveries on the replication mechanism and the genetic structure of viruses. Born in Turin, Italy, into an influential Jewish family, he attended the medical school at the University of Turin following which he served as a medical doctor in the Italian army for some time. He then proceeded to study radiology at the University of Rome where he developed an interest in bacteriophages (viruses that infect bacteria).

Device "scale" Scales (or weighing scales) are devices to measure weight. Spring balances or spring scales calculate weight that is the product of mass into gravity (9.807 m/s2) on the force on a spring, whereas a balance or pair of scales using a balance beam compares masses by balancing the weight due to the mass of an object against the weight of one or more known masses. The balance or pair of scales using a traditional balance beam to compare masses may read correctly for mass even if moved to a place with a different non-zero gravitational field strength. An analytical balance (often called a "lab balance") is a class of balance designed to measure small mass in the submilligram range. The measuring pan of an analytical balance (0.1 mg or better) is inside a transparent enclosure with doors Laboratory Methods, Level 3

so that dust does not collect and so any air currents in the room do not affect the balance's operation. Types of balance Analytical balances are designed to measure small masses from around 320g to sub-milligram. They are very sensitive pieces of equipment so need to be treated with care. The main types of laboratory balance are (masses stated are general values only): 1. 2. 3. 4.

Top-pan balance (200g – 0.001g) Analytical balance (320g – 0.0001g) Microbalance (6g – 0.000001g) Ultra-microbalance (6g – 0.0000001g)

Explain the technique that works on this device? ………………………………………………… ………………………………………………… ………………………………………………… ………………………………………………… ………………………………………………… References: https://www.thefamouspeople.com/profiles/salvador-luria-7398.php https://www.ncbi.nlm.nih.gov/pmc/articles/PMC392330/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC431765/

Some tools and Devices in biotechnology laboratories Tool or Devices Laboratory Methods, Level 3

description

picture

Droppers

These are small glass tubes with narrow tips on one end and a rubber bulb on the other. They suck up liquid that can then be squeezed out in small drops. These can be used to add an indicator to a solution about to be titrated Dry Block designed for applications that Heaters require repeatable results and superior temperature stability. These units are designed for applications such as incubation and activation of cultures, enzyme reactions, immunoassays, melting/boiling points, and a wide variety of other laboratory procedures. Dispenser Laboratory dispensers provide a simple means of storing and dispensing liquid and other materials in the laboratory. A lab dispenser varies in available volume rang from as small as 0.2 microliters up to 100 milliliters, depending on the model Dissecting low magnification microscope microscope used especially for examining or dissecting biological specimens (viewing the surface features of specimen not tissues or cells). Invented and developed by Philip O. Gravelle. Features: dissecting microscopes have two eyepieces that provide binocular vision that allow the user to see objects in 3D.

Laboratory Methods, Level 3

DNA sequencers

a DNA sequencer is used to determine the order of the four bases: G (guanine), C (cytosine), A (adenine) and T (thymine). This is then reported as a text string, called a read.

Water distiller

Distillation is the process of separating the components or substances from a liquid mixture by selective boiling and condensation. Distillation may result in essentially complete separation (nearly pure components), or it may be a partial separation that increases the concentration of selected components of the mixture. In either case the process exploits differences in the volatility of the mixture's components. In industrial chemistry, distillation is a unit operation of practically universal importance, but it is a physical separation process and not a chemical reaction. It is a Laboratory method used in the field of microbiology to transfer and inoculate living microorganisms.

Needle (Inoculation Needle)

Nano-Fil Syringe

Nano-Fil is a specially designed syringe developed in response to customer requests for improved microinjection in mice and other small animals. It makes quantitative nanoliter injection much easier and more accurate than any other method currently in use. Retinal pigment

Laboratory Methods, Level 3

epithelium injection Mouse brain injection Nebulizer A nebulizer is a device that turns liquid medicine into a mist. As you breathe, the mist of medicine moves into your lungs. The medicine may be an antibiotic or other medicine for your lungs. Nano liter It is a flat plate with multiple Plate wells used as small test tubes. It is a miniaturized version of the microliter plates that are standard tools in analytical research.

Napco Vacuum Oven

It's used for a variety of applications like drying heat treatment of materials under vacuum conditions in industrial and mining enterprises, laboratories, medical colleges, and scientific research institutes. is specially designed for drying of material which is thermosensitive or decompounds and oxidative easily. It can be filled with inert gases to the inner chamber, which is especially for a rapid drying of some compound material. Vacuum oven temperature range RT+10 Â°C ~ +250 Â°C, capacities ranging from 20 liters to 210 liters

Nitric Oxide WPI offers the most extensive Sensors range of nitric oxide (NO) sensors available. Developed over a decade of extensive research in the field of nitric oxide, the result is a superior range of NO sensors Laboratory Methods, Level 3

Watch glass

Wash-bottle

that enable routine detection of nitric oxide at ultralow concentrations. Selectivity of WPIâ&#x20AC;&#x2122;s Nitric Oxide Sensors The ideal nitric oxide sensor should be insensitive to other reactive species likely to be present within the measurement environment. The conventional Nafion coated carbon fiber nitric oxide sensor exhibits a large response to such species. WPIâ&#x20AC;&#x2122;s unique nitric oxide sensor technology utilizes a novel surface membrane which amplifies the response to NO while eliminating responses to a vast range of reactive species, including nitrite, absorbic acid, hydrogen peroxide, catecholamines, and much more. A watch glass is just a round piece of glass that is slightly concave/convex It can hold a small amount of liquid or solid. They can be used for evaporation purposes and can function as a lid for a beaker. A wash bottle is a squeeze bottle with a nozzle, used to rinse various pieces of laboratory glassware, such as test tubes and round bottom flasks.

Laboratory Methods, Level 3

Warburg apparatus

A device for gauging the oxygen consumption and carbon dioxide production of incubated tissue slices by manometric measurement of changes in gas pressure produced by oxygen absorption in an enclosed flask.

Laboratory Methods, Level 3