Biotechnology Basics (written by Sandra Slivka, Ph. …faculty.sdmiramar.edu/bhaidar/Bio 133/BiotechnologyBasics 08.pdf · • the use of microorganisms or cultured plant and animal

“Biotechnology Basics” prepared for Bio 133 San Diego Miramar College Author: Sandra Slivka Ph.D.

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Biotechnology Basics (written by Sandra Slivka, Ph. D.)

Topics Covered Background Information

What is Biotechnology? What are the Kinds of Jobs? What is GMP/GLP? What is the FDA? Intellectual Property Laboratory Notebooks

Basic Lab Skills

Lab Safety Measurements, Accuracy and Precision Reading and Understanding Protocols Solutions Dilutions Spectrophotometry


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What is Biotechnology? Although the term biotechnology is a term that recently came into everyday language, mankind has actually been employing biotechnology throughout history. Biotechnology is any technology which employs biological systems (e.g. cells or organisms) or components of cells and organisms (e.g. enzymes, antibodies) to develop a process and achieve an applied goal. Therefore, both making bread and medical research rely on biotechnology. From this general concept of biotechnology, three appropriate definitions for biotechnology can be derived. Biotechnology is: • the industrial application of biological, biomedical or biochemical discoveries • the use of microorganisms or cultured plant and animal cells as factories for the production

of bio-chemicals • the use of cells or cellular components and by-products by industry In prehistoric times, mankind developed biotechnology processes to ferment beverages (e.g. wine and beer), make vinegar, bread, cheese, yogurt, and sauerkraut. Not much changed until the 1870’s when Louis Pasteur improved both beer and wine technologies. Pasteur also developed rabies and chicken cholera vaccines. In the twentieth century, industrial fermentation techniques were perfected. Fermentation refers to large-scale microbial reactions to obtain microbial byproducts. In fermentation, microbes are the industrial machines. Acetone and butyl alcohol have been produced by advanced fermentation techniques. Microbial factories that produce antibiotics, such as penicillin, were created. More advancement came with mammalian cell culture. For example, polio vaccine was produced by mammalian cell culture. These advancements gave mankind clues how to harness cells and microorganisms as biological factories. Modern biotechnology began in the 1970’s with a modern understanding of DNA and genetics. Today, the biotechnology industry combines this understanding of genetics and genetic engineering with the ancient fermentation sciences. Genetic engineering is a technique that uses recombinant or cloned genetic material (DNA) and places it into an organism that does not normally express that material. By adding genetic material to microorganisms, cells can be used as factories to produce important modern biotechnology products. These products are proteins and antibodies, the core products of today’s biotechnology industry. The biotechnology industry has applied its discoveries to a wide variety of fields. In agriculture, biotechnology has led to crop improvement, increased milk production, and insect resistance of plants. The environment has benefited via genetically engineered organisms that degrade sewage and industrial wastes. Scientists in the industry are now engineering ethanol-producing microbes to yield large quantities of ethanol from low cost renewable resources such as cellulose. Some biotechnology products are pharmaceuticals such as antibodies or recombinant vaccines. Biotechnology techniques have been applied to pathogen detection. Molecular probes have been developed to rapidly detect species and strains of infecting organisms. In medicine, the industry is now exploring the use of modern techniques for genetic therapies.


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Shown in this figure is a common rod shaped bacterium (e.g. Escherichia coli) which can, after receiving DNA from a recombinant gene, become a factory that produces a protein for human use. Techniques employed in making this organism into a factory for recombinant protein will be discussed in this course.

Homework: Visit www.biocomworkforce.org and view the “What is Biotechnology” Series. Answer these questions. 1. What are three general applications of biotechnology? 2. Use your text and/or the internet to find an example of each one. 3. Do you have any ethical concerns about these applications?


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What Types of Jobs Exist in Biotechnology? Most biotechnology companies start with an idea. From this idea, the laboratory methods must be developed to make this idea into a product. This is the research phase of Research and Development (R&D). Once the research is complete, development of the idea into a product takes several steps including manufacturing and clinical trials. This process can be broken down into three phases of growth for a biotechnology company: • Start-up • R & D Only • Production and R & D During the start-up stage, a biotechnology company establishes its legal structure, submits proposals for grants or funding, and begins preliminary research. During this stage the employment is largely research scientists. During the R & D stage, the company must determine what its product will be and how it will take it to market. The R & D stage may also include clinical trials. Clinical trials involve producing larger quantities of product for testing, resulting in a more diverse and larger staff. Clinical trials require interaction with the medical community and the FDA (Food and Drug Administration). In this stage, the Regulatory Affairs Department begins to grow. Production in addition to R & D is the last stage of growth. As a biotechnology company matures and product refinement takes place, manufacturing increases. In this stage, diverse employment in manufacturing and the support of manufacturing is necessary. Similarly, Sales and Marketing Departments also become necessary. At this stage, employment to support manufacturing in production jobs, production support, quality control and assurance, and sales and marketing increases. The company will also work to develop other products to increase its product line. This is done with additional research. To support these three stages of growth, a company’s employment profile inevitably changes. This is not only requires growth in the number of employees, but expansion of the variety of positions that need to be filled. These changes are summarized in the following table.


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EMPLOYMENT AT VARIOUS STAGES OF GROWTH OF A COMPANY STAGE OF GROWTH TYPE OF EMPLOYMENT Research (Very Early) Discovery Research Ph.D. Research Scientists A.S./B.A. Research Assistants/Associates Efficacy in Disease Animal Technicians Development Stage Testing of Product Pilot Manufacturing Clinical Trial Support Process Development FDA Approval Regulatory Affairs Marketing and Sales Production of Protein Manufacturing Quality Control Quality Control (QC) jobs Marketing Sales and Technical support


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GMP and GLP Overview Good Laboratory Practices (GLP) for Non-clinical Laboratory Studies is often employed in the biotechnology industry. Quality control labs MUST use GLP. GMP (Good Manufacturing Practices) applies to factories and other manufacturing facilities. The regulations for both GLP and GMP are similar. Documentation is the key to both GLP and GMP. According to ALL regulatory agencies, if it is not written down and documented, it was not done! In this class – if you did not record it in your lab book, it was not done!

Requirements for both GLP and GMP are in place to: o Maintain test and control specimen identity o Protect specimens from contamination o Assure that protocol instructions are followed and documented. In practice, there are three important aspects of quality control to insure that good laboratory practices are followed. The three aspects are: the "quality" of the personnel, the equipment, and the chemical reagents. These are the three aspects that comprise a "quality" product or test. Below is an outline or practices which assure the "quality" of these three aspects of quality control. I. Personnel A. Adequate training for task B. Certified record of training C. Following protocols (Standard Operating Procedures (SOPS)) D. Record keeping proves that SOPs have been followed II. Equipment A. Personnel are trained on how to properly use the equipment B. Equipment is adequately maintained (record of maintenance) C. Equipment is monitored to be sure it is properly functioning


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D. Equipment is validated/calibrated at regular intervals III. Chemical Reagents A. Reagents have been checked for purity and accuracy B. Maintenance of Lot Records C. Vendor Audits In both GLP and GMP, stringent documentation is in place. The purpose of this documentation is to allow complete traceability of all reagents used and results of all experiments. Complete traceability allows determination of the source of any product or experimental failure. For more information about GMP, go to http://www.gmp1st.com/index.htm Homework: The Food and Drug Administration (FDA) 1. Take a virtual tour of the FDA http://www.eduneering.com/fda/courses/fdatour/welcome.html

What do they regulate? What did you learn that surprises you? 2. History of the FDA Use these links to help you http://www.fda.gov/oc/history/default.htm http://www.fda.gov/oc/history/slideshow/default.htm .

• Prepare a timeline for the major milestones in food regulation? Why were they important?

1898 Tea Act Protect consumers against inferior tea 1906 U.S. Pure Food and Drug Act 1938 1962 1997 Any thing else you think is impt?


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Drug Discovery Then and Now

Visit the website and Answer the Following Questions: 1. What is an IND? http://www.fda.gov/cder/Regulatory/applications/ind_page_1.htm 2. What is a NDA? http://www.fda.gov/cder/Regulatory/applications/NDA.htm 3. There are evolving expectations of the FDA outlined below.

• Traditional Expectations for the Drug Regulatory System: – All marketed drugs are effective and safe within the context of their use. – Human drugs are of high quality. – Generic competition keeps drug prices reasonable. – All advertising and promotion of drugs is informative and is not false or

misleading. http://www.havidol.com/


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• Evolving Expectations for the Drug Regulatory System: – Patients who lack alternatives have access to investigational drugs. – High-quality information about how to use drugs is available, including

information on children, elderly patients, and other groups. – Robust drug development programs that thoroughly protect human subjects

flourish and are productive What did you learn from the website about these evolving expectations? The FDA Regulates the Manufacture of New Drugs to Insure Efficacy and Safety. Please explain how the FDA insures Efficacy? Safety?


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Intellectual Property Copyrights and Patents Adapted from http://people.howstuffworks.com/patent1.htm There is an established system for protecting intellectual property, the product of a person or company's originality and creativity. The broadest protection of this sort is the copyright. Copyrights are intended to protect "original works of authorship" that are in a tangible form. This includes paintings, books, movies, choreographed dances (if the steps are written down), music, architecture and all other sorts of art. For a set length of time, these works cannot be copied or reproduced without the copyright-holder's permission. In the United States, the protection extends for the life of the copyright-holder plus 70 years (for works created after January 1, 1978). If a company owns the copyright, the protection lasts anywhere from 95 to 120 years depending on whether or not the work was published. When inventors come up with a new device or discovery, the first thing they want to do is patent it. Patents are a government's way of giving an inventor ownership of his or her creation. For a certain period of time, patent-holders are allowed to control how their inventions are used, allowing them to reap the financial rewards of their work. Patents are a palpable, legally-binding manifestation of a person's genius and innovation; they allow a person to actually own an idea. Of all of the forms of intellectual-property protection, patents are the most complex and tightly regulated. Patents are basically copyrights for inventions, defined by U.S. patent law as "any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof." Unlike copyrights, patents protect the idea or design of the invention, rather than the tangible form of the invention itself. Consequently, patenting something is a much trickier procedure than copyrighting something. Inventions must meet three criteria: They must be novel, non-obvious and useful. To patent an invention, you have to meet a number of requirements. First of all, the invention must be sufficiently novel. That is, it must be substantially unlike anything that is already patented, has already been on the market or has been written about in a publication. In fact, you can't even patent your own invention if it has been on the market or discussed in publications for more than a year. Adaptations of earlier inventions can be patented as long as they are non-obvious, meaning that a person of standard skill in the area of study wouldn't automatically come up with the same idea upon examining the existing invention. For example, you can't patent the concept of making a toaster that can handle more pieces of bread at once, because that is only taking an existing invention and making it bigger. For an invention to be patented, it must be innovative to the point that it wouldn't be obvious to others. Another condition for patenting something is that the invention is "useful." Generally speaking, this means that the invention serves some purpose and that it actually works. You couldn't patent a random configuration of gears, for example, if it didn't do anything in particular. You also wouldn't be able to patent a time machine if you couldn't construct a working model.


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Unproven ideas generally fall into the realm of science fiction, and so are protected only by copyright law. The "useful" clause may also be interpreted as a prohibition against inventions that can only be used for illegal and/or immoral practices. All a patent really does is give the patent-holder the right to stop others from producing, selling or using his or her invention. For the life of the patent (20 years in the United States), patent-holders can profit from their inventions by going into business for themselves or licensing the use of their invention to other companies. It is up to the patent-holder to actually enforce the patent; the government does not go after patent or copyright infringers. To enlist the government's help in stopping infringement, the patent-holder must take any infringers to court.

Should Genes be Patented? Some sorts of ideas are considered outside the realm of patents. No matter how innovative and beneficial they may be, certain notions are automatically public property the minute they are uncovered. The most prevalent examples of this are discoveries in the natural world. Scientists cannot patent laws of the universe, even though defining those laws may revolutionize a particular industry or change how we live. Einstein's Law of Relativity, for example, revolutionized the world of physics and will be forever linked with the man who

devised it, but it has never been owned by anybody. This principle existed long before humans did, so, logically, it cannot be any person's intellectual property. Scientists cannot patent a newly discovered plant or animal, either, though they may be able to patent a new plant or animal that was produced through genetic engineering. This is similar to the patenting of processes and computer programs: A genetic engineer didn't create any of the parts, but the combination of these parts may be novel and non-obvious, and therefore patentable. Patents help encourage the advancement of science and technology. Patents do this in two major ways:

• They give inventors an opportunity to profit from their creations. The process of inventing a new device or process is an extremely difficult one, and few people would go through it if there weren't any financial reward.

• They help disseminate technological information to other inventors. When you apply for a patent, you are required to submit a detailed description of your invention. This description becomes part of the patent office's database, which is public record. Once the patent has expired, the idea is more readily available than it would have been if it had never been patented.

Patents motivate individual inventors, but they also motivate large companies. They are particularly important to chemical, computer-technology and pharmaceutical firms. In these


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markets, your success might be wholly dependent on having exclusive rights to innovative products. Intellectual property makes up a huge chunk of these companies' assets. In biotechnology, it might be the entire assets! When something is invented as part of a person's work for a company, the company is typically given control over the invention, though the patent may officially go to the individual inventor. This arrangement varies depending on the country and the nature of the employee's contract. If you are contracted to grant your employer all patent rights to your work, selling your own invention would actually be infringing your own patent (and your employer could take you to court). The same holds for copyrighted "work-for-hire." You may be the original creator, but if you republish the work yourself, you are infringing the copyright. Patenting an idea is usually a long, expensive and difficult process. There are two main types of patent professionals: patent lawyers and patent agents. Patent lawyers are attorneys with a science or technical degree who have met the patent office's qualifications (their professional credentials have been reviewed and they have passed a qualifying test). Patent agents are people who have met the patent office's qualifications but are not recognized as attorneys. Some inventors work through the patent process themselves (called working pro se), but most hire a patent lawyer or patent agent early on in the process. Steps in obtaining a patent: 1) Patent Search: check to make sure that no one has patented it before

Optional Assignment: Think up a crazy invention. Go to http://www.google.com/patents and see if it exists.

Were you surprised? Explain

2) File a patent application The application is made up of a number of different parts. It must include: • A list and description of any "prior art," earlier inventions that are relevant to your

invention. • A brief summary outlining the new invention • A description of the "preferred embodiment" of the invention. This is a detailed account

of how your idea will actually be put into practice. One or more "claims." Claims are the most important element of the application, as they are the actual legal description of your invention. Down the road, if you need to take someone to court for infringing on your idea, the strength of your suit will largely depend on your claims. The patent lawyer has the necessary training to ensure that your claims provide the highest legal protection.

3) Once the examiner is satisfied with your application, you are issued a "Notice of

Allowance." All you have to do at this point is pay the patent fees including periodic maintenance fees, which come out to thousands of dollars. But if your idea is good enough, the reasoning goes, you will make much more money by licensing your patented idea. If it’s really good, then you will want to pursue patenting in every industrialized country to protect your invention and your income world-wide.


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The Laboratory Notebook

Confirming experimental evidence is much like recreating prizewinning chocolate chip cookies. Both are much easier when a written record is available. While laboratory notebooks often contain recipes for experiments, they serve several other functions as well. Scientists maintain notebooks to refer to previous experimental procedures and data. Notebooks allow scientist to reproduce protocols and results without starting over. Imagine that each time you make chocolate chip cookies, you needed to determine the amount of sugar, flour, etc to add!

Notebooks are crucial documents that establish intellectual property ownership. Intellectual property is the main asset of biotechnology companies. Intellectual property ownership can be established in a court or the legal system using properly maintained laboratory notebooks. Thus, notebooks are legal documents that are dated, signed and witnessed to prove progress, invention, and having met an objective. In general, academic scientists are far less rigorous about keeping a notebook. On the other hand, industrial scientist standards are more rigorous. At companies, notebooks are typically duplicated, witnessed, and kept under lock and key. The pages are numbered and the notebooks are not spiral or loose-leaf, but bound like books. Witness and researcher signatures are keys to a legally sound notebook. A witness provides corroboration from someone who understands and works in the field. All company lab notebooks are hardbound notebooks kept in pen. These notebooks should remain in the lab at all times. Primary research data should be part of the notebook. Electronic data can be referred to but must be guarded electronically from manipulation. Notebook contents should include: data, background information, safety instructions, objectives, lists of materials and methods, and results and conclusions. Raw data is important. If you use a paper towel or the cuff of a sleeve to record data, you must put it in your notebook. An important rule is to write data while it is fresh. You can look it over later from a different perspective. Standard format should be used whenever possible. As a student, it is useful to write a Pre-lab outline in your notebook. Most scientists do this prior to an experiment. Everything that is done in the lab should be documented and written in your notebook. Also important is to write everything that does not happen as expected. Finally, you must keep all results, analyses, and graphs and tables. The most important test of a good notebook is if the experiments can be easily reproduced. Notebooks are crucial in intellectual property disputes. In the US, the first to invent must show proof. Validation of results and protecting intellectual property are not the only uses of laboratory notebooks. When one scientist takes over for another, a situation which happens frequently, the value of a well kept notebook is obvious. A lab notebook is more than a personal diary- it is an important part of doing science!


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Guidelines for Keeping a Laboratory Notebook

1. Use only a bound notebook with number pages. Do not rip out pages!

2. Make all entries ink (preferably black!).

3. Make your entries legible, clear and complete.

4. All observations and data must be entered immediately and directly into your notebook.

5. Cross out errors with a single line. Date and initial all cross-outs. Text below a

cross-out must be legible. Never erase!

6. Note all problems. Never try to obscure, erase, or ignore a mistake. Be honest!

7. Blank lines and unused pages should be crossed out with a single line. This prevents tampering with the notebook.

8. Make observations that are as objective as possible, stating what you observed,

not what you interpret!

9. Always include detailed information about instruments, reagents, samples, materials, and equipment used. Make sure it is possible to account for and trace all materials used.

10. Be certain that the laboratory notebook is stored in a secure location.

Practice your empty page cross-out here!


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Lab Safety Guidelines • Personal Protective Equipment (PPE): Use laboratory coats, safety glasses

and gloves as appropriate. Avoid restrictive clothing and open-toed shoes. • No Eating, Drinking or Smoking in the Laboratory. • No Mouth Pipetting. • Universal Safety Precautions: When using potentially

biohazardous materials work in a sanitary manner and treat all waste as a potential biohazard.

• When using potentially hazardous chemicals become

educated to the risks and use appropriate care in handling and disposal.

• Before working in the laboratory learn the locations of the eye wash, shower,

safety blanket, and telephone with emergency numbers. • Fire Hazards: Note the location of fire extinguishers. Use extra care when

flammable liquids such as ethanol are used. • Disposal: Dispose of petri dishes, pipette tips and all other materials according

to instructions given by laboratory supervisors. • Decontamination: Any item potentially contaminated by

microorganisms should be treated with 70% alcohol. I agree to abide by these safety practices ________________________________________ _________ Signature Date


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Measurement of Volume with Pipettes Pipetting is an essential technique in the laboratory. A pipette is a calibrated device that takes up a liquid by suction. Accurate volume measurement is necessary to assure that protocols are properly followed. To measure small volumes in the microliter (µl) and milliliter (ml) range, a variety of pipettes are used:

A. Micropipettors- The volume range of digital micropipettors varies depending on the manufacturer. Ranges include small volume (0.5-10 µl or 1-20 µl) or large volumes with a range of 100-1000 µl.

B. Transfer Pipettes- Small Polypropylene transfer pipettes are handy because they have an integrated bulb.

C. Pasteur Pipettes - Glass pipettes for which an external bulb is needed. D. Standard Pipettes- Plastic disposable or glass pipettes with marked volume gradations for

accurate measurements in the milliliter range. Common pipette sizes are available in 1, 5, 10, and 25 ml sizes. An external bulb or electronic pipet-aid is required for suction.

plunger (operates with thumbscrew to adjust

volume setting

barrel- do not immerse

disposable pipette tip

tip ejector mechanism

THE DIGITAL MICROPIPETTOR

THE PASTEUR PIPETTE


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Use of Standard Serological Pipettes Pre-sterilized, disposable pipettes (1, 5, 10 or 25 ml calibrated) are convenient and supplied bulk with many sterile pipettes to a package, or individually wrapped. Non-sterile glass or plastic pipettes are used when sterility is not a concern. To dispense bulk-packed sterile pipettes:

• Open immediately before use

• Cut one corner of the plastic wrapper at the end opposite the pipette tips. Avoid touching and contaminating the wrapper opening

• Tap bag to push the pipette end through the cut opening. Pull the pipette out of the bag, touching only the top of the pipette near the cotton plug.

• Affix the top end into the pipet-aid or bulb and proceed with dispensing liquid.

• Close bag with tape to keep sterile for future use.

To use individually wrapped pipettes properly: • Peel back only enough of the wrapper to expose the top, wide end of the pipette and

affix end into the pipet-aid or bulb. Completely peel back the wrapper immediately before use.

Use of Digital Micropipettors Never! Never! Never! (Do I have to say it again?)

1. Never rotate volume adjustor beyond the upper or lower range of the pipette.

2. Never use pipettor without tip in place: this could ruin the precision piston that measures the volume of fluid.

3. Never lay down pipettor with filled tip; fluid could run back into piston.

4. Never let plunger snap back after withdrawing or ejecting fluid: this could damage

piston.

5. Never immerse barrel of pipettor in fluid.

6. Never flame pipettor. General Micropipettor Directions

1. Rotate volume adjustor to desired setting. Note change in plunger length as volume is changed. Be sure to locate decimal point properly when reading volume setting.

2. Firmly seat proper-sized tip on end of micropipettor.

3. When withdrawing or expelling fluid, always hold tube firmly between thumb and forefinger. Hold tube at nearly eye level to observe the change in the fluid level in pipette tip. Do not pipette with tube in test tube rack or have another person hold tube while pipetting.

4. Each tube must be held in the hand during each manipulation. Grasping the tube body rather than the lid provides more control and avoids contamination from the hands.


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5. Hold pipettor almost vertically when filling.

6. Most digital micropipettors have a two-position plunger with resistance “stops”. These resistance stops can be felt with the thumb. Depressing to the first stop measures the preset volume. Depressing to the second stop introduces additional air to expel any solution remaining in the tip.

7. To withdraw sample from a tube filled with reagent: o depress plunger to first stop o dip tip into solution o slowly release plunger o slide tip along side of tube (removes solution on outside of tip) o check that tip is properly filled (no air on bottom or bubbles)

8. To expel sample into reaction tube: o touch pipette to inside wall of tube into which the sample is to be delivered o slowly depress plunger to the first stop to expel sample. o depress to second stop to blow out last bit of sample. o remove pipette from tube while holding plunger down to avoid sucking up sample o eject tip into waste beaker on lab bench


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Lab Exercise: Pipettor Training and Documentation in a GMP/GLP environment Perform this exercise in groups of 4. The most experienced person in the group performs the exercise first. Document your work as you would on a routine basis in a GLP/GMP environment by initialing and dating all steps performed. Student number 2 performs the exercise with the same pipette set. Student 1 witnesses the ‘training’ and documentation skills of person number 2. This training is also documented in this exercise. Finally, students 1 and 2 proceed to train students 3 and 4 on the use of the pipettors. I. Large Volume Micropipettor Exercise This step is to practice use of the 100-1000 µl micropipettor. Errors are more common with this pipettor because if the plunger is not released slowly while withdrawing solutions, an air bubble may form or solution may be drawn into the piston. 1. Use a marker to label two 1.5 ml reaction tubes A and B. 2. Use the matrix below as a checklist while adding solutions to each reaction tube. Add

samples as follows while avoiding cross-contamination. Initial and date to document that reagents have been added:

a. Using a fresh tip, add solution I to tubes A and B. b. Using a fresh tip, add solution II to tubes A and B. c. Using a fresh tip, add solution III to tubes A and B. d. Using a fresh tip, add solution IV to tubes A and B.

To avoid sample cross contamination in pipetting:

o always add appropriate amounts of single reagent to all reaction tubes o release each reagent drop on a new location on inside wall of tube so the same tip

can be used to pipette into many tubes o use fresh tip for each new reagent to be transferred o if the tip becomes contaminated, switch to a new one.

Important - Initial and date each addition below! Matrix Checklist for Adding Solutions Initial and date observations! Tube Sol I Sol II Sol III Sol IV Initial and Date A 100 µl 200 µl 150 µl 550 µl

B 150 µl 250 µl 350 µl 250 µl

A total of 1000 μl of reagent was added to each tube above. To check that the measurements were accurate, set micropipettor to 1000 μl and carefully withdraw solution from each tube. Record what you observe on checking the pipetting (tip just filled, small volume left in tube, air space in tip etc.)


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Record your observations below Initial and date observations! Tube Observations A

B

If necessary, repeat this exercise to obtain proper volumes. II. Small Volume Pipettor Exercise- This simulates a reaction setup using a micropipettor with a range of 0.5-10 µl or 1-20 µl. 1. Mark three microfuge tubes A, B, and C 2. Use the matrix below as a checklist while adding solutions to each reaction tube. Add

samples in same way as in the large-volume micropipetting exercise to avoid cross-contamination. Initial and date after each addition to document that reagents have been added:

Important - Initial and date each addition below! (Note: These are the Amgen Solutions Matrix Checklist for Adding Solutions Initial and date observations!

Tube dH2O Solution 1 Solution 2 Solution 3 Total volume A 2 μL 4 μL ⎯ 4 μL 10 μL B 2 μL ⎯ 8 μL ⎯ 10 μL C 2 μL ⎯ ⎯ 8 μL 10 μL

3. Close tube caps. Pool and mix by centrifuging in microcentrifuge. 4. BE SURE THAT THE CENTRIFUGE IS BALANCED

5. A total of 10 µl of reagent was added to each tube

above. To check that the measurements are accurate, set pipette to 10 µL and very carefully withdraw solution for each tube. Is tip just filled or under filled? Is any solution left in the tube? Record what you observe on checking the pipetting.


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Record your observations below Initial and date observations! Tube Observations

Significant Figures Research in biology requires frequent measurement of mass and volume. It is important to record these measurements in a manner that allows the data to be understood correctly. For example, you might weigh two objects and obtain measurements of 0.24 g and 0.23 g. You may need to determine if these numbers are significantly different. This is done using significant figures. Significant Figures are all of the digits known with certainty PLUS the first digit whose value is estimated. Significant Figures are used because: 1. Every measurement in the laboratory has a “degree of certainty”. The certainty is often

determined by the resolution of the device we are using.

2. Reporting data to other scientists means that we are also telling them our “degree of certainty” as the accuracy, precision, and resolution of the data.

3. Using and understanding significant figures allows scientists to communicate accurately. Exercise: Significant Figures A biotechnology company specifies that the level of RNA impurities in their gene therapy DNA must be less than or equal to 0.02%. The quality control lab measures the RNA in a particular lot to be 0.024%. Does this lot of product meet specifications?


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2Mathematical Manipulations with Significant Figures It is very important to understand that when you perform a mathematical operation using several measurements, the final value can have no more significant figures that the measurement with the least number of significant figures. For example, the concentration of a solution is calculated as mass/volume. If you added 3.42 mg of sucrose to 10.00 ml of water, the concentration would be 3.42 mg/10.00 ml= 0.342 mg/ml. The volume is recorded to 4 significant figures and the weight is expressed in 3 significant figures. Therefore, there may only be 3 significant figures in the final expression of concentration. Exercise: Mathematical Manipulations with Significant Figures You pipette 2.0 ml of standard into a tube. You then add 0.125 µl of reagents. How do you express the final volume of liquid in the tube? Measurements in the Lab Measurement of Weight: The Equipment A variety of balances may be used in the biology lab. There are generally two types:

o Analytical Balances for weighing masses from 1 mg to 10 g. These balances are delicate and must be used with care.

o Top-loading electronic balances or triple beam mechanical balances for weighing

masses from 10 mg to several hundred grams. Rules for using balances correctly:

o Never place material to be measured directly on the pan. Objects to be weighed are placed on weighing paper or in weigh boats or pans.

o Many balances have a tare feature that will allow you to set the balance to zero after the

weigh paper or pan is placed on the balance. This makes it unnecessary to subtract the mass of the weighing container from the total mass.

Measurement of Volume: The Equipment Many kinds of devices are used to measure volume. 1. Micropipettes - These are used with a disposable tip. Use them only in the range that they

are calibrated for. 2. Glass or plastic pipettes - These come in a number of forms. Note whether they are

calibrated to the tip (some are not). You use a pipetting device such as a “Pipet Aid” to


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aspirate and dispense volumes with a pipette. Insert the pipette firmly in the pipetting aid. Handle only the top of the pipette. The pipette graduations should be facing you. Use the meniscus of liquid bottom to accurately read the volume. While holding the container containing the liquid to aspirate, insert the pipette into the solution at an angle. Fill the pipette with slightly more that the desired final volume and expel the last drop of excess fluid. Touch the tip of the pipette to the container wall to remove excess liquid. Move the pipette to the receiving container and expel the desired volume of fluid, touching the tip against the wall to remove any fluid outside the pipette. If possible, the fluid should be expelled directly into a solution already present in the receiving container.

3. Graduated cylinders - These measure volumes in the range of 10 ml to 5000 ml. Use the

best graduated cylinder for the desired volume. These are filled by adding fluid until the bottom of the meniscus is level with the desired graduation on the side of the cylinder. For accuracy, perform measurements at eye-level.

4. Beakers and flasks - Although beakers and flasks often have graduations on them, these

are not very accurate. For many solutions this approximation of volume is acceptable. Rules for Measuring Volumes 1. Volumes are usually measured to three or four significant figures, depending on the

measuring device. A general rule to follow is that the volume measured should, if possible, be equal to at least 1/3 of the capacity of the device. The reasons for this are two-fold: 1) most devices must usually be filled 10-20% before it is possible to measure to 3 significant figures and 2) the impact of a small error in filling or delivery will be reduced if the total volume delivered is larger.

2. For devices such a pipetteman (micropipettor) that are regulated by air pressure, do not

draw too rapidly. If you do, you risk air bubbles and inaccurate measurement of viscous liquids.

3. Be careful to ensure that there is no drop of fluid adhering to the outside of a pipette. This

could cause a significant error in measurement. Absorbent paper such as ‘Kimwipes’ are often used to wipe off tips. If using this technique, be sure that the paper does not draw fluid from inside the device.

4. Expel the content of the pipette near the bottom of a receiving container. It is ideal to

dispense into a larger volume of fluid so that there is no loss on the inside of the container. As the volume delivered becomes smaller, this becomes more important.

Accuracy and Precision It’s the only way to know if the product is the same! Accuracy and Precision Accuracy is an estimation of how closely a value represents the true value. Accuracy is determined by comparison to a validated, external standard. The closer the value obtained in an assay to the validated standard, the more accurate the measurement.


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Precision is the reproducibility of the measurement. Precision is determined statistically by evaluating the standard deviation of the measurements. The smaller the standard deviation, the more precise the measurement! Consider an arrow and a target. Accuracy is hitting the bulls-eye. Precision is hitting the same spot over and over again. Lack of precision and accuracy comes from various types of error: 1. Operational or Procedural error. There are operator errors that improve with practice but never disappear because we are human! 2. Systematic error. This is from malfunction of measuring devices. 3. Random variability. Biological systems are variable. When working with living things there is inherent variability. Exercise: Accuracy and precision. What’s the difference? On the two targets below show 6 accurate measurements that are not precise compared to 6 accurate measurements that are very precise.


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On the next two targets, show 6 precise measurements that are not accurate compared to 6 precise measurements that are very accurate.

Exercise: Accuracy and Precision Question: Is it better to use a P-1000 or a P-200 for measuring (200 µl)? Review “How to use Balances” on page 23, above. NOW…You can answer this question by determining Accuracy and Precision 1. Each student works individually 2. Place a weigh boat on a top-loading balance and tare it. 3. Remove 200 µl of water from a beaker and weigh. 4. Record your measurement. Do NOT empty the weigh boat. 5. Tare the balance again. 6. Repeat steps 3-5 four more times. 7. Repeat steps 1-6 using a 1 ml pipette (measuring (200 µl) P 200 P 1000

Measurement Weight(g) Weight(g) addition 1 addition 2 addition 3 addition 4


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addition 5 Average Standard Deviation Coefficient of Variation

The accuracy of your measurements can be evaluated by knowing that 1 ml of water should have a mass of 0.99823 g at 20°C. The precision can be determined by looking at the standard deviation (SD). If the SD is divided by the mean x 100 (a percentage also known as the coefficient of variation) is <10% than you measurement are precise. If time permits, repeat the above experiment using 1 ml volumes. Compare a 1 ml pipette, 5 ml pipette, Pasteur pipette (20 drops) and a transfer pipette. Use Excel to Determine Accuracy and Precision: Discuss below if your measurements were accurate and precise. If not, discuss the types of error that may have contributed to this lack of accuracy or precision. How did you determine accuracy and precision using Excel? Lack of precision and accuracy results from various types of errors: 1. Operational or procedural error. There are operator errors that improve with practice but never

disappear because we are human!

2. Systematic error. This is caused by measuring device malfunctions.

3. Random variability. Biological systems are variable. When working with living things, there is inherent variability.

The standard deviation (SD) is a measurement of how “spread out” the data is from the mean value and is the most calculated measure of variability in a data set. A low standard deviation means that the data are tightly clustered; a high standard deviation means that they are widely scattered. Assuming that the data set is normally distributed as shown to the right (bell curve):

• approximately 68% of all data points will fall within (+/-) 1 standard deviation from the mean; • approximately 95% of all data points will fall within (+/-) 2 standard deviations from the mean; • approximately 99% of all data points will fall within (+/-) 3 standard deviations from the mean.

Data points within 2 standard deviations are considered statistically significant. Data that are 3 standard deviations or more from the mean are “outliers” and are considered insignificant or invalid. Standard deviation can be calculated by hand, or through the use of a statistical calculator or an excel spreadsheet. The coefficient of variation is the SD divided by the mean x 100%. If this value is <10%, then your measurement is precise. Using your data, discuss below if your measurements were accurate and precise. If not, discuss the types of error that may have contributed to this lack of accuracy or precision.


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Excel in the Laboratory Basic built-in functions. (AVERAGE, MEAN, MODE, COUNT, MAX, MIN) Adapted from: http://phoenix.phys.clemson.edu/tutorials/excel/stats.html We will use the familiar example of a class's grades to illustrate the use of some of the more basic Excel functions, like AVERAGE( ), MODE( ) AND MAX( ). Assume a class's grade distribution is as follows: 3, 0, 4, 4, 4, 2, 4, 1, 4, 0, 3, 3, 1, 1, 3. These grades are based on a 4-point scale with 4=A and 0=F and are entered into an Excel worksheet shown below. Using the AVERAGE( ) function, we find the class's average (or arithmetic mean) grade is a disappointing 2.47, or a mid-C. The syntax for this common function is =AVERAGE(number1, number2, ...) and is displayed in the screen shot below. However we don't get a clear picture of the class's performance by simply looking at its average. We can further analyze the data using the MEDIAN( ) function. The median gives the middle number in a set of numbers and its syntax is =MEDIAN(number1, number2,...). We see from the screen shot that the median grade is 3.0, meaning that half of the grades are higher than 3.0, and half are lower. Therefore, despite the low class average, more students scored 3's and 4's than 2's, 1's and 0's. Additionally, we can also analyze the grade distribution by using the MODE( ) function. The mode gives the most frequently occurring value of a set of numbers and its syntax is =MODE(number1, number2,...). From the screen shot, we see that the mode grade is 4, meaning that a score of 4 was the most common grade. Again, the instructor of the class can take heart that, despite the low class average, more students made A's than any other grade. Without going into too much detail, we can also use some of Excel's built-in functions to determine the number of grades entered, and the maximum and minimum grades of the


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distribution. The syntax for these functions are shown below in the list and also in the screen shot.

• The COUNT( ) function gives the number of cells that contain numbers. Its syntax is =COUNT(value1, value2, ...).

• The MAX( ) function returns the largest value in a set of numbers. Its syntax is =MAX(number1, number2, ...).

• The MIN( ) function returns the smallest value in a set of numbers. Its syntax is =MIN(number1, number2, ...).

Error analysis tools. (STDEV) AND COEFFICIENT OF VARIATION Let's assume we make a number of repetitive measurements of one quantity, say the weight of 1 ml of water. Assuming our balance is accurate, water should weigh 1 gm per ml. We can take the average of repeated measurements. This will tell us the ACCURACY of our measurement. (You will need to determine the percent error from the accepted value) But this tells us nothing of the precision of our measurement. For this, we need to calculate the standard deviation of the measured values. To quickly determine the standard deviation of any measurement, use Excel's built-in STDEV( ) function. In Biotechnology Standard Deviation is usually used to determine a Coefficient of Variation as follows: =(stdev/mean)/100. A Coefficient of Variation of variation of 10% is ideal! Why do you think that this is used instead of Stdev? Standard Curves: Excel Can Be used to plot a standard curve. The independent variable (what you already ‘know’) about the sample should be on the X-axis. The dependent variable (what you are measuring) should be on the Y-axis. Sample Data Standards Concentration mg/ml Absorbance nm 0.00 0.000 0.15 0.004 0.30 0.012 0.60 0.027 1.25 0.055 2.50 0.096 5.00 0.348 10.00 0.815

Unknown Absorbance 0.013


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Procedure: 1. Begin by opening your graphing program. 2. In cell A1 write absorbance. 3. In cell B1 write concentration. 4. Type the data into the two columns. 5. Highlight all of the cells which you have put information into. 6. Click on Insert and then click on Chart. 7. Click on the XY (Scatter) option. Check to see that the picture that is

high-lighted does not have any lines connecting the points. 8. Click on Next. 9. Check to see that the points are on the sample graph. Click Next. 10. Type Standard Curve in the blank under chart title. 11. Type Absorbance (nm) in the blank under Value (X) Axis. 12. Type Concentration (mM) in the blank under Value (Y) Axis. 13. Click on Next. 14. Click on the circle next to As New Sheet. 15. Click on Finish. 16. Click on the name of the graph. A box will come up. Click on the Font tab. Find

the word underline and click on the pull down box to highlight single. Click on OK. This will underline the title of your graph.

17. Click on Chart and then on Add Trendline. 18. Click on the Type tab. Be sure the picture for the Linear Trendline is

high-lighted. Click on the Options tab and click the box to the left of Display Equation on Chart. This will place a check in this box. Click on OK.

19. You will see the equation for the line at the top of the Graph. This is the equation you will need to calculate the concentrations from the absorbances you recorded on the spectrophotometer. Copy down this equation below.

Results and Analysis 1. The equation for the line is _____________________________________ 2. Find the concentration for the unknown absorbance. Show all of your work. (Hint: your absorbance is the “X” in this equation.)


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Solutions, Dilutions, and Colorimetric Assays Percent Solutions and Concentration Solutions In the biotechnology laboratory, solutions are made with required specifications. Percent Solutions and Concentration Solutions are the language that describes reagents for experiments. To calculate the amount of material to prepare certain solutions, equations are routinely used. These equations will be reviewed here. Percent Solutions tells us the number of parts per 100. A percent solution can be weight/volume or volume/volume (w/v or v/v). Concentration is the amount of a particular substance in a stated volume (or sometimes mass) of a solution or mixture. The substance that is dissolved is called the solute. The liquid in which the solute is dissolved is called the solvent. A molar solution is the number of moles/liter. Moles are determined by the molecular weight. Using Equations to Prepare Solutions Use equations to mathematically determine the amount needed. One side of the equation should state what is known and the other should have the equivalent that you need to know. Homework: 1. A laboratory solution is made of water and ethylene glycol. How could you prepare 250 ml

of a 30% ethylene glycol solution?

What do you need to know? You need to know how many ml of 100% ethylene glycol is needed for a 30% solution (250 ml) _________ ml 100% ethylene glycol = 250 ml x 30% ethylene glycol Another way to express this equation is V1C1 =V2C2. If you state this in words, you can ask how many ml (V1) of ethylene glycol at its initial concentration of 100% (C1) does it take to make 250 ml (V2) of 30% ethylene glycol (C2). This is a proportionality relationship. Solve this equation here.

2. If a solution requires a concentration of 3 g of NaCl in 250 ml total volume, how much NaCl

is required to make 1000 ml?

You know that you need 3 g/250 ml. How many grams do you need for 1000 ml? 3 g/250 ml= X/ 1000 ml. (Hint: this is also a proportional relationship). Solve this problem here.


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3. If you were asked to calculate the molarity or moles/liter (M) of the solution you just prepared

in the previous question what do you need to know? 4. Calculate the molarity (M) of the solution you prepared. Formula or Molecular weight of the

NaCl is 58.44 daltons or grams. 1M X X g (answer to question 2 above) = ? M 58.44 g

Solve this problem here.

5. Now, you are asked to prepare 100 ml of 2M NaCl solution. How much NaCl do you weigh

to dissolve in 100 ml of distilled water? First, calculate how many grams you need to make 1000 ml. 58.44 g X 2M = ? g/1000 ml (answer a) 1M Next, Use the same proportional relationship in question #2 to calculate the weight in grams

needed to make 100 ml instead of 1000ml. answer a above = ? . 1000 ml 100 ml

Dilutions Dilutions are used frequently in the biotechnology lab. Dilution errors account for the majority of many of the mistakes. Dilution terminology is often inconsistent among scientists: clarify when necessary based on what you know. Dilution terminology based on American Society of Microbiologists Recommendations 1. 1 part combined with 9 part means that there is 1 part in 10 ml total volume or 1/10 2. An undiluted substance, by definitions, is called 1/1. 3. When talking about dilution the symbol : (colon) means parts. Examples: A 1:2 dilution means there are three parts total volume A 1/2 dilution means there are two parts total volume A dilution series is a group of solutions that have the same components but at different concentrations.


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• An independent dilution series is one where each dilution is independent of the others.

• A dependent or serial dilution is on where all have the same dilution factor (for example all are 1/10 or 1/2).

The concentration of a diluted solution in the final tube is determined by multiplying the concentration of the original solution times the dilution in the first tube, times the dilution in the second tube and so on until reaching the last tube. The concentration of a diluted solution is determined by multiplying the concentration of the original solution times the dilution factor (expressed as a fraction). Example: In a protein assay, the amount of protein in 1 ml of diluted sample was 87 mg. If the original sample was diluted 1/50, what was the concentration of the original sample? 87 mg/ ml = x mg/ml x1/ 50 Solve here for the protein concentration in the original sample. Colorimetric Assays Biotechnologists are often interested in determining the concentrations of substances in solutions. For example, a biotechnologist might want to know the concentrations of glucose in blood or urine if they are looking for the effect of an insulin-like drug on diabetics. They might measure protein quantity to determine the amount of a recombinant product. A method for measuring something is called an assay. Colorimetric assays use the light absorbing properties of molecules to indicate quantity. The amount of light that a compound absorbs can be used to determine the concentration of the compound present in solution. Such concentration dependent methods are called colorimetric assays. The general goal of a colorimetric assay is to make sure that the substance of interest is the only substance that is able to absorb light of a given wavelength. The typical assay compares the absorption of a test substance termed the “unknown” with the absorption of a previously determined amount of the “known” substance. The absorbance of various amounts of the known are determined and a standard curve is constructed. The amount of substance is plotted graphically on the x-axis and the absorbance of light of a specific wavelength on the y-axis. This is the standard curve. Using the standard curve, the biotechnologist can determine the amount of substance in the unknown. Colorimetric assays work in part because of a basic physics principle called “Beer’s Law”. Beer’s Law states that if a substance that absorbs light is dissolved in a transparent solvent (like water), then the absorbance of the solution is proportional to the concentration of the absorbing substance. Beer’s Law is not valid at all concentrations. Measurements in colorimetric assays can only be made in the concentration range of the amount of substance that is proportional to the light absorbance. This is usually the same as the linear range of the assay.


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Exercise: Serial Dilution and Colorimetric Assay Prepare serial dilutions Materials Required: DNP-glycine 5 mM standard solution (known) made in diluent 0.5 ml per students DNP-glycine (unknowns) 1 ml per student 0.1 M NaOH (diluent) 60 ml per student. Each student works individually. This is a ‘test’ of the skills you practiced this week! 1. Obtain 21 16x120 mm test tubes. Place these in a rack. 2. Label three of these tubes 1/2, 1/4 and 1/8. Prepare serial 2-fold dilutions of the unknown

DNP-glycine solution. You want to have 0.5 ml final solution of the three dilutions. How will you prepare these? Following preparation of the serial dilutions you will have four unknown tubes; three 1/2 serial dilutions and the undiluted standard. You prepared these serial dilutions so that at least one of your unknown tubes falls in the range of the standard curve. These tubes are your “unknowns”.

3. Label 10 tubes 1-10. In these tubes, you will use the known solution to create the standard curve. Use the protocol in the table below to create the standard curve. This curve will be run in duplicate.

4. Calculate the nmoles of DNP-glycine per tube with the class and place the results in the table. Use the following sample calculation: 10µl (known) x 5 mmoles/liter = (10 x 10 -6 liters) x (5 x10-3 moles/liter) = 50 x 10-9 moles = 50 nmoles Note how useful scientific notation was to this problem solution. Also, note how important it is to keep your units consistent!

Tube # µl diluent µl standard DNP-glycine nmoles standard/tube

1 2

100 100

0 0

3 4

90 90

10 10

5 6

80 80

20 20

7 8

50 50

50 50

9 10

0 0

100 100


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5. Label 7 more tubes 11-18. Add 100 µl of unknown prepared in step 2 to each of these tubes as follows: Tube # Dilution___ 11 undiluted 12 undiluted 13 1/2 14 1/2 15 1/4 16 1/4 17 1/8 18 1/8

Question: Why are you using 100 μl of each of these? 6. Add 3 ml of 0.1 M NaOH to each tube from 1-18. 7. Set your spectrophotometer to a 410 nm absorbance. Perform the necessary calibration of

the instrument. Blank the spectrophotometer to tube #1. 8. Read the absorbance of each tube and record the reading in the table below. Be sure to

wipe the outside of the tube before reading. Why did you blank to tube #1?

Why are you wiping each tube prior to reading?

Tube (Stds)

A Tube (Stds)

A Tube (Unknowns)

A Tube (Unknowns)

A

1 6 11 16 2 7 12 17 3 8 13 18 4 9 14 5 10 15

9. Graph your standard curve (absorbance vs. concentration) and calculate the amount

of unknown in your original solution. Attach your graph and show your calculations for the concentration of your unknowns here.

Here is the equation you will need for solving your unknown concentrations: amount (nmoles) = concentration in nmoles x mmol x 106 µL = mmol = mM 100 µl* µl 106 nmoles L L * Why 100 µl? This equation works for the undiluted sample. What will you need to do to determine the concentration in the diluted samples?


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If time permits, talk about solution preparation. Students often have trouble doing basic math. Some examples as following. a. If you were asked to make 200ml of 2M NaCl, how many grams of NaCl do you need to weigh? b. If you were asked to prepare 500ml of 0.5M NaCl, how many mls of 2M NaCl do you need to use? c. If you were asked to prepare 300ml 0.9% NaCl, how many grams of NaCl do you need to weigh?


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Appendix: Solutions and Dilutions Solutions made using percentage by weight (w/v) The number of grams in 100mL of solution is indicated by the percentage. For example, a 1% solution has one gram of solid dissolved in 100mL of solvent. To make this type of solution properly, you should weight 1g and dissolve it in slightly less than 100mL. Once the solids have dissolved, you can bring the volume up to the final 100mL (see animation below). Solutions made using percentage by volume (v/v) In this case, the percentage indicates the volume of the full strength solution in 100mL of dilute solution. Molar Solutions A 1 molar solution is a solution in which 1 mole of a compound is dissolved in a total volume of 1 litre. For example: The molecular weight of sodium chloride (NaCl) is 58.44, so one gram molecular weight (= 1 mole) is 58.44g. If you dissolve 58.44g of NaCl in a final volume of 1 litre, you have made a 1M NaCl solution. To make a 0.1M NaCl solution, you could weigh 5.844g of NaCl and dissolve it in 1 litre of water; OR 0.5844g of NaCl in 100mL of water; OR make a 1:10 dilution of a 1M sample. Many of the solutions you will use are described in terms of their molarity, so check that you are comforable with the concept by describing how you would make 500mL of a 0.05M NaCl solution pH of a Solution The pH of a solution is a measure of its acidity. The pH is defined as the negative log of the hydrogen ion concentration. The pH scale ranges from 0 to 14 where 0 is the most acidic, 14 is the most basic, and 7 is neutral. Remember that since pH is a log scale, the difference between any two numbers is a factor of 10, so precise measurement of pH is very important. In the lab, pH can be measured with a pH meter or pH paper. Buffered Solutions A buffered solution resists changes in pH. Buffers are used when tissues or organisms are to be maintained at a precise pH, usually between pH 7.2 and 7.4 for human and many animal tissues.


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A phosphate buffer is one of the more common buffers used and is made up of a precise mixture of sodium monobasic phosphate (NaH2PO4) and sodium dibasic phosphate (Na2HPO4). Dilutions: Sometimes it is necessary to use one solution to make a specific amount of a more dilute solution. To do this, you can use the formula: V1C1 = V2C2 where: V1 = volume of starting solution needed to make the new solution C1 = concentration of starting solution V2 = final volume of new solution C2 = final concentration of new solution For example: Make 5mL of a 0.25M solution from 2.5mL of a 1M solution. V1C1 = V2C2 (V1)(1M) = (5mL)(0.25M) V1 = [(5mL)(0.25M)] / (1M) V1 = 1.25mL So you will need to use 1.25mL of the 1M solution. Since you want the diluted solution to have a final volume of 5mL, you will need to add ( V1-V2 = 5mL - 1.25mL) 3.75mL of diluent. Dilution Problems 1. a. How would you make 10mL of a 1:10 dilution of a 1M NaCl solution? b. What would the final concentration of NaCl be from 1a above? c. How would you make 80mL of a 1:20 dilution of a 1M NaCl solution? d. How would you make 50mL of a 1:25 dilutions of a 1M NaCl solution? 2. How would you prepare exactly 6mL of a 1/20 dilution (assume the concentration of your starting solution is "1")? 3. You are provided with an antibody solution (Ab) that has a concentration of 600 microgram (μg) / microlitre (μL). For lab, it is necessary make the following dilutions: a. 10μL of 600μg/μL Ab + 190μL of buffer to make a 1:20 dilution at _____μg/μL


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b. 20μL of 1:20 Ab + 40μL of buffer to make a 1:60 dilution at _____μg/μL c. 5μL of 1:60 Ab + 5μL of buffer to make a _____dilution at _____μg/μL d. 10μL of 1:60 Ab + 90μL of buffer to make a_____dilution at _____μg/μL e. 10μL of 1:60 Ab + 40μL of buffer to make a_____dilution at _____μg/μL f. 10μL of 1:60 Ab + 10μL of buffer to make a_____dilution at _____μg/μL For Serial Dilutions animation go to: www.wellesley.edu/Biology/Concepts/Html/serialdilutions.html


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Introduction to Biotechnology Vocabulary Assay-Any method for determining the presence or quantity of a component; Qualitative or quantitative analysis of a substance, especially of an ore or drug, to determine its components Autoclave - A strong, pressurized, steam-heated vessel, as for laboratory experiments, sterilization, or cooking. v : subject to the action of an autoclave Caustic-capable of burning, corroding, dissolving or eating away with chemical action Colorimetric-An instrument that measures concentration of a known constituent of a solution by comparison with colors of standard solutions of that constituent Hybridoma-a clone of hybrid cells produced by fusing an antibody-producing cell with a cancerous cell that grows will in culture Antibody:a protein formed in the blood to fight against the disease (very specific!) Monoclonal-A homologous population of antibodies produced by a hybridoma Polyclonal-A variety of produces of many different clones of lymphocytes Recombinant-A DNA molecule created by joining two or more DNA fragments from two or more sources Mutagenic-An agent, such as a chemical, ultraviolet light, or a radioactive element, that can induce or increase the frequency of mutation in an organism. Buffer- Substance that minimizes change in the acidity of a solution when an acid or base is added to the solution. Biotechnology- Use of microorganisms, such as bacteria or yeast or biological substances, such as enzymes. Applications include the production of certain drugs, synthetic hormones. mutagenic: a substance that is capable of increasing the risk of a mutation inert: having few or no active properties labile: unstable, capable of changing Mole- The amount of a substance that contains as many atoms, molecules, ions, or other elementary units as the number of atoms in 0.012 kilogram of carbon 12. The number is 6.0225 × 1023, or Avogadro's number. Solvent- substance in which another substance is dissolved, forming a solution or a substance, usually a liquid, capable of dissolving another substance Aseptic- Free of pathogenic microorganisms: aseptic surgical instruments. b. Using methods to protect against infection by pathogenic microorganisms


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Biohazard- A biological agent, such as an infectious microorganism, or a condition that constitutes a threat to humans, especially in biological research or experimentation Mole- The amount of a substance that contains as many atoms, molecules, ions, or other elementary units as the number of atoms in 0.012 kilogram of carbon 12. The number is 6.0225 × 1023, or Avogadro's number. Molar solutions - number of moles of substance in a total of 1 liter of solvent. % weight/volume - number of grams of substance per 100 ml of a solvent. % volume/volume - number of milliliters of a solution in a combined total of 100 ml of solvent. Enzyme - any of numerous proteins or produced by living organisms and functioning as biochemical catalysts Catalyst- a substance organic (enzyme) or inorganic (iron etc.) that speeds up a reaction without being consumed in the reaction Meticulous: extremely careful and precise, in details Elucidated – To make plain, especially by explanation; clarify Reagent: a substance used to cause a chemical reaction, specially to detect the presence of another substance. Carcinogen: any substance that causes cancer. clone – n. The aggregate of the asexually produced progeny of an individual; also: a group of replicas of all or part of a macromolecule (as DNA or an antibody). An individual grown from a single somatic cell of its parent and is genetically identical to it. One that appears to be a copy of an original form. Etymology: Greek klOn twig, slip; akin to Greek klan to break (Date: 1903) in vi·tro - adv. & adj. in an artificial environment outside the living organism: an egg fertilized in vitro; in vitro fertilization. New Latin in vitro: Latin in, in + Latin vitro, ablative of vitrum, glass. In vivo- in a living organism pep·tide - n. any of various natural or synthetic compounds containing two or more amino acids linked by the carboxyl group of one amino acid to the amino group of another. A peptide is any compound produced by amide formation between a carboxyl group of one amino acid and an amino group of another. The amide bonds in peptides may be called peptide bonds. The word peptide usually applies to compounds whose amide bonds are formed between C-l of one amino acid and N-2 of another (sometimes called eupeptide bonds), but it includes compounds with residues linked by other amide bonds (sometimes called isopeptide bonds). Peptides with fewer than about 10-20 residues may also be called oligopeptides; those with more, polypeptides. Polypeptides of specific sequence of more than about 50 residues are usually known as proteins, but authors differ greatly on where they start using this term.

Documents

Biotechnology Basics (written by Sandra Slivka, Ph. …faculty.sdmiramar.edu/bhaidar/Bio 133/BiotechnologyBasics 08.pdf · • the use of microorganisms or cultured plant and animal