A key component of biotechnology education is development of technical skills that take your students out of the books and into the laboratory. Confused on how to start setting up your lab? Let us help! Edvotek was founded in 1987 as the first company focused on translating cutting-edge biotechnology for the teaching classroom. We work with educators all over the world to demystify science and foster the next generation of scientists through hands-on, active learning activities. That is why we are happy to provide you with this FREE guide, full of experiments and technical skills tutorials to help you set up your biotechnology lab. We hope you enjoy teaching and learning with Edvotek!
EDVOTEK® • THE BIOTECHNOLOGY EDUCATION COMPANY®
EDVOTEK® BIOTECHNOLOGY BOOTCAMP
Edvotek ® Biotechnology Bootcamp
A key component of biotechnology education is development of technical skills that take your students out of the books and into the laboratory. Confused on how to start setting up your lab? Let us help! Edvotek was founded in 1987 as the first company focused on translating cutting-edge biotechnology for the teaching classroom. We work with educators all over the world to demystify science and foster the next generation of scientists through hands-on, active learning activities. That is why we are happy to provide you with this FREE guide, full of experiments and technical skills tutorials to help you set up your biotechnology lab.
We hope you enjoy teaching and learning with Edvotek!
Table of Contents
Page
Biotechnology Lab Basics
3 5 7 9
Micropipetting
Restriction Enzymes Structure of DNA
Agarose Gel Electrophoresis and Gel Interpretation Bacterial Transformation and Transformation Efficiency
11 13 20 24 27
ELISA
Polymerase Chain Reaction - PCR
Proteins and SDS-PAGE
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Biotechnology Lab Basics
ARTICLES & VIDEOS ABOUT BASIC LABORATORY SKILLS:
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Biotechnology Lab Basics
ARTICLES & VIDEOS ABOUT LABORATORY SAFETY:
RESOURCES ABOUT BIOTECHNOLOGY CAREERS:
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Micropipetting
Pipettes are essential laboratory tools used by scien - tists to measure and manipulate liquids. They range from simple eye dropper-like devices with squeezable bulbs to advanced robotic systems capable of precise volume dispensing. Micropipettes , in particular, play a crucial role in accurately measuring small volumes, ensuring successful and reproducible experiments.
In biotechnology labs, where reactions involve micro- liter volumes, scientists rely on piston displacement micropipettes. The accuracy of pipetting is of utmost importance in these experiments, as even minor incon- sistencies like air bubbles or extra droplets can disrupt reaction proportions. Therefore, to optimize your stu- dents’ laboratory results, it is critical to make sure that they have mastered the technical skill of micropipetting.
MICROPIPETTING EXPERIMENTS:
VIDEOS ABOUT MICROPIPETTING:
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Micropipetting
ARTICLES ABOUT MICROPIPETTING:
MICROPIPETTING QUICK GUIDE:
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Restriction Enzymes
One of the most significant discoveries in molecular biology belongs to a group of enzymes called restric - tion endonucleases, also referred to as restriction enzymes . These remarkable enzymes function like molecular scissors by precisely cleaving through the sugar-phosphate DNA backbone based on the spe-
cific sequence of nucleotides. The immense utility of restriction enzymes revolutionized numerous scientific endeavors, including molecular cloning, DNA mapping, sequencing, and a wide array of genome-wide studies. This discovery launched the era of modern molecular biotechnology.
RESTRICTION DIGEST EXPERIMENTS:
Image Attribution: Helixitta, CC BY-SA 4.0 <https://creativecommons.org/licenses/by-sa/4.0>, via Wikimedia Commons
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Restriction Enzymes
ARTICLES ABOUT RESTRICTION DIGESTS:
VIDEOS ABOUT RESTRICTION DIGESTS:
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Structure of DNA
Choose different colors for each component and then color in the worksheet.
Base pair
5'
3'
T
A
S
Nitrogen Bases
Color
S
Base pair
Adenine (purine)
A
Guanine (purine)
C
G
G
S
Cytosine (pyrimidine)
C
S
Thymine (pyrimidine)
T
C
Color
Sugar-Phosphate Backbone
G
S
deoxyribose (sugar)
S
S
Hydrogen bonds
phosphate
Nucleotide
T
A
Phosphate
Nitrogen base
S
S
Sugar
Hydrogen bonds 5’ = Five prime end 3’ = Three prime end
C
G
S
S
T
A
S
S
C
G
S
S
3'
5'
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Structure of DNA
CREATE A 3-D MODEL OF DNA Students can work in groups of 2-4. REQUIRED MATERIALS: • Toothpicks (wood or plastic) • Bag of multicolored soft candy (Gumdrops, gummy bears, or marshmallows work well. Must have four colors) • Bag of licorice sticks • Digital Camera or Cell Phone (optional) PROCEDURE: 1. If students intend to eat the candy after the experiment, be sure they thoroughly clean their hands before creating the model. Plastic wrap or aluminum foil can be placed over desktops to create a clean surface. 2. Each group will receive the following items in a plastic bag: a. Four colors of gummy candy represent the nucleotide bases. Each group should receive at least six candies of each color. Assign a nucleotide to each of the four colors of gummy candy. i. Adenine =_____________________ ii. Thymine =_____________________ iii. Cytosine =_____________________ iv. Guanine =_____________________ b. Licorice sticks represent the sugar-phosphate back- bone. Each group should receive two. c. Toothpicks represent the base pairing interactions between nucleotides. Each group should receive at least toothpicks. 3. Using a toothpick, skewer two of the gummy candies. Be sure to follow base pairing rules (A=T, G=C). Repeat until all of the gummy candy is used. 4. Attach one piece of licorice to each side of the toothpick. The base pair toothpicks should be added to the licorice in random order. The resulting DNA duplex should look like a ladder, with the licorice as the rails and the nucleo- tides as the rungs. 5. Pick up the ladder at each end. Carefully twist the DNA model so that it forms a double helix.
ASSESSMENT:
The instructor should evaluate each DNA model before the students consume the candy. Alternatively, students pho- tograph their final products for submission. The photo is printed, the component parts of DNA are labeled, and the photo is entered into the student’s lab notebook.
DISCUSSION QUESTION: Record the base pair sequence of your DNA molecule. Why is the order of the nucleotides important?
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Agarose Gel Electrophoresis and Gel Interpretation
Agarose gel electrophoresis is a biotechnology technique that uses electricity and a porous gel matrix to separate mixtures of molecules based on their charge, shape, and size. It is commonly used to separate dyes, proteins, and nucleic acids like DNA
and RNA. In particular, it is especially useful in analyz- ing mixtures of DNA fragments generated by restriction enzyme digestion. During agarose gel electrophoresis, these DNA fragments are separated into distinct bands according to their respective sizes.
DNA ELECTROPHORESIS EXPERIMENTS:
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Agarose Gel Electrophoresis and Gel Interpretation
ARTICLES & VIDEOS ABOUT ELECTROPHORESIS:
ELECTROPHORESIS INQUIRY GUIDE:
https://www.edvotek.com/site/pdf/02_Electrophoresis_inquiry_guide.pdf
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Bacterial Transformation and Transformation Efficiency
heat shock transformation , the cells are treated with calcium chloride to make them “competent”. DNA is added to the cells before they are moved quickly be - tween two very different temperatures. It is believed that the combination of calcium chloride and the rapid change in temperature changes the permeability of the cell wall and membrane, allowing DNA molecules to enter the cell. In practice, transformation is highly inefficient—only one in every 10,000 cells successfully incorporates the plasmid DNA. However, since many cells are used in a transformation experiment (about a billion cells), only a few cells must be transformed to achieve a positive outcome. We can use the data from our experiment to determine how well our transformation worked.
In nature, non-essential genes are found on small circu- lar pieces of double-stranded DNA known as plasmids . These plasmids facilitate the exchange of beneficial genes among bacteria and can replicate independently from the cell’s chromosomal DNA. In the lab, we can modify plasmids by introducing genes from various sources. When these engineered plasmids are intro - duced into bacteria, they transform the bacteria into liv- ing factories capable of producing valuable substances like medications, vitamins, and insulin, which is used to treat diabetes. Since E. coli are not naturally competent, we need to force them to take up plasmid DNA in the lab. This can be done with electricity, in a process called electropora- tion, or through physical means in a heat shock. In a
BACTERIAL TRANSFORMATION EXPERIMENTS:
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Bacterial Transformation and Transformation Efficiency
ARTICLES ABOUT TRANSFORMATION:
VIDEOS ABOUT TRANSFORMATION:
TRANSFORMATION TROUBLESHOOTING GUIDE:
https://www.edvotek.com/site/pdf/Transformation_Troubleshoot.pdf
TRANSFORMATION INQUIRY GUIDE:
https://www.edvotek.com/site/pdf/03_Transformation_inquiry_guide.pdf
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Bacterial Transformation and Transformation Efficiency
CALCULATING TRANSFORMATION EFFICIENCY
The basic unit of all living organisms, from bacteria to humans, is the cell. Most cells contain DNA, which is the genetic blueprint used to build an organism. In nature, bacteria pass small pieces of DNA back and forth through transformation. In the laboratory, we can force cells to take up DNA using the “heat shock” technique, where the combination of charged ions and a rapid change in temperature force bacteria to take up DNA from the surrounding environment. In practice, transformation is highly inefficient-only one in every 10,000 cells successfully incorporates the plasmid DNA. However, since many cells are used in a transformation experiment (about a billion cells), only a few cells must be transformed to achieve a positive outcome. We can use the data from our experiment to determine how well our transformation worked by calculating the transformation efficiency. This is a quantitative determination of the number of cells transformed per 1 µg of plasmid DNA. In essence, it is an indicator of the success of the transformation experiment. To calculate the transformation efficiency: Count the number of colonies on the transformation plate. A convenient method to keep track of counted colonies is to mark each colony with a lab marking pen on the outside of the plate.
Determine the transformation efficiency using the following formula:
Number of transformants
Final vol. at recovery (mL) vol. plated (mL)
Number of transformants per µg
=
Number of transformants
Final vol. at recovery (mL) vol. plated (mL)
X
Number of transformants per µg
µg of DNA
=
X
Example: Assume you observed 40 colonies:
µg of DNA
40 transformants
1600 (1.6 x 10 3 ) transformants per µg
Transformation efficiency generally ranges from 1 x 10 4 to 1 x 10 8 cells transformed per µg plasmid. A fun way to explore this concept in class would be to change the heat shock conditions and to analyze the results. Can you make your transformation more efficient by adding more DNA or changing the duration or temperature of the heat shock? Try it and find out! 40 transformants 0.5 mL 0.05 µg 0.25 mL 1600 (1.6 x 10 3 ) transformants per µg X =
0.5 mL
=
X
0.05 µg
0.25 mL
Practice problem for calculating transformation efficiency:
The bacterial plate, at right, represents the results from your most recent transformation experiment. In this experiment, you added 15 ng of DNA to 125 µL of competent cells for transformation. After heat shock, 300 µL of nutrient broth was added to the cells for recov- ery. 100 µL of the cell suspension was plated on the selective agar plates before they were incubated overnight. Using the data from your transformation experiment, calculate the efficiency.
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Bacterial Transformation and Transformation Efficiency
SOURCE PLATES: THE FOUNDATION TO A SUCCESSFUL TRANSFORMATION!
Over the years, we have improved our protocols to provide consistent results. We h ave found that a well-streaked source plate is one of the most important parts in guaranteeing a successful transformation!
What is a Source Plate?
A source plate is a standard LB Agar plate that is used to grow the initial colonies of bacteria that will be used for the transformation process. Source plates allow scientists to observe the growth of the bacteria to make sure it is grow-ing well and there is no contamination. Because of the way source plates are streaked, students are able to select well-isolated colonies to get the proper amount of bacteria for their transformation! We have found that when the source plates are older than 24 hours, and when clumps of bacteria are used rather than individual colonies, transformation is far less likely to succeed. Always pick the well-isolated colonies on your plate that are 1-1.5 mm in diameter.
Source Plates Should Be:
• Prepared the day before the lab and incubated overnight at 37ºC. • Used as soon as possible after the incubation period (18-22 hours) has finished. • Streaked properly to ensure the growth of well-isolated colonies (see figure, below).
FIGURE 1
Cover.
90°
90°
90°
Invert & incubate.
Add BactoBead™.
Rehydrate.
Streak 1st quadrant.
Rotate. Streak 2nd quadrant.
Rotate. Streak 3rd quadrant.
Rotate. Isolate colonies.
Streaking Source Plates:
1. Add a fresh BactoBead™ to an LB Agar plate. 2. Rehydrate the BactoBead™ with 10 µL of recovery broth. 3. Start a dense patch of bacteria in the first quadrant by spreading the BactoBead™ back in forth. 4. Rotate the plate 90º and drag the loop through the bacteria in the first quadrant. Streak back and forth making sure not to dip back into the previous quadrant. 5. Repeat this on the 3rd quadrant.
6. Drag the loop across the rest of the empty space on the agar to isolate colonies. 7. Cover and invert the plates, then place them in a 37ºC incubator overnight.
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Bacterial Transformation and Transformation Efficiency
TIPS FOR MAKING SOURCE PLATES:
• You can practice this technique by using a marker on the base of the plate and mimicking the steps in Figure 1. • Your students can practice this too! Pass out pieces of paper or white boards and have your students master streaking. • Ensure the agar is fresh and not dried out. Avoid puncturing the plate with the inoculation loop when streaking the bacteria. • Incubate the plate inverted at 37ºC overnight. • Make sure that the BactoBead™ has been stored correctly: Keep the vial capped tightly and in the refrigerator, as exposure to air and moisture will ruin the BactoBead™. • Make sure the BactoBead™ is rehydrated properly with 10 μL recovery broth.
Watch our instructional video here:
All our instructional videos can be found at youtube.com/edvotekinc
After the incubation:
After the overnight incubation has finished it is important to check on the status of your source plates. The source plate should have isolated colonies that are about 1-1.5 mm in diameter, all the same color, and the agar should not be dried out or hard.
Picking colonies:
When it is time to perform the transformation, students will want to pick up between 5-10 well-isolated colonies for the experiment. To do this, provide the students with sterile inoculation loops or toothpicks and gently swipe the colonies to get them adhered to the loop or toothpick. Then these colonies will be resuspended in ice cold calcium chloride to make them competent! Make sure your students are gentle, and do not pierce the agar with the loop or toothpick.
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Bacterial Transformation and Transformation Efficiency
STORY TILES: TRANSFORMATION Below are story tiles depicting transformation in bacterial cells. CUT OUT the cards and REORDER them in the correct sequence. FILL IN the numerical sequence in the left box of each story tile for future reference or PASTE onto a new sheet of paper in the correct order.
X
X
X
X
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Bacterial Transformation and Transformation Efficiency
STORY TILES: TRANSFORMATION PROTOCOL Below are story tiles depicting a common lab procedure for creating competent cells (bacteria cells that can take in exogenous DNA). Using the lab instructions provided below as a guide, CUT OUT the cards and REORDER them in the correct sequence. FILL IN the numerical sequence in the left box of each story tile for future reference or PASTE onto a new sheet of paper in the correct order.
plasmid solution
42°C
Approx. 5 colonies
1. ADD Calcium Chloride to a sterile test tube and place on ice for 2 minutes. 2. Using a sterile loop, TRANSFER 5 well isolated bacteria colonies to the test tube containing Calcium Chloride.
3. TWIST the loop between your fingers to free the cells. 4. RESUSPEND the bacteria cells by pipetting up and down. 5. ADD the plasmid solution to the tube. 6. INCUBATE on ice for 10 minutes. Just one tube. 7. TRANSFER to a warm water bath, incubate for 45 seconds.
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ELISA
The Enzyme-Linked Immunosorbent Assay , or ELISA , is a highly sensitive test that uses antibodies to detect the presence of specific molecules within a complex sample. is an invaluable tool for researchers, as it can identify even trace amounts of antigens in biological samples, making it ideal for pathogen and allergen
detection, among other applications. The fundamental principle of ELISA revolves around the use of antibod - ies to identify antigens present in the samples. With the capability to generate antibodies for a wide range of molecules, ELISA has emerged as a versatile laboratory test.
ELISA EXPERIMENTS:
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ELISA
ARTICLES ABOUT ELISA:
VIDEOS ABOUT ELISA:
ELISA INQUIRY GUIDE:
https://www.edvotek.com/site/pdf/01_ELISA_inquiry_guide.pdf
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ELISA
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ELISA
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Polymerase Chain Reaction (PCR)
In the early 1980’s, Kary Mullis developed a technique that replicated DNA in vitro using short, synthetic DNA oligonucleotides designed to target a specific se- quence (known as primers) and DNA Polymerase I. In a process similar to replication in a cell’s nucleus, the primers would bind to the DNA, directing polymerase to copy the gene sequence. However, after the initial elongation, the sample was heated to denature the newly-formed DNA duplex, then cooled to allow prim-
er binding and extension to happen again. Each time the sample cycled through the different temperatures, the amount of DNA doubled. By repeating this cycle of heating and cooling many times, billions of copies of a specific DNA sequence were produced in a matter of minutes. This simple cycle – anneal, extend, denature – is the basis of the polymerase chain reaction . Be - cause of its ease of use and its ability to rapidly amplify DNA, PCR has become indispensable in the medical and life sciences lab.
PCR EXPERIMENTS:
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Polymerase Chain Reaction (PCR)
ARTICLES ABOUT PCR:
VIDEOS ABOUT PCR:
PCR TROUBLESHOOTING GUIDES:
https://www.edvotek.com/site/pdf/PCR_Electrophoresis_Troubleshoot.pdf and https://www.edvotek.com/site/pdf/Human_PCR_Troubleshoot.pdf
PCR INQUIRY GUIDE:
https://www.edvotek.com/site/pdf/04_PCR_inquiry_guide.pdf
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Polymerase Chain Reaction (PCR)
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STORY TILES: PCR Below are story tiles depicting a single cycle in a PCR Reaction. CUT OUT the cards and REORDER them in the correct sequence. FILL IN the numerical sequence in the left box of each story tile for future reference or PASTE onto a new sheet of paper in the correct order.
primer
primer
dNTP
dNTP
DNA
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Proteins and SDS-PAGE
SDS polyacrylamide-gel electrophoresis, or SDS-PAGE, is a technique that is used to separate proteins according to their molecular weight. Proteins produce a unique challenge for electrophoresis because they have complex shapes and different charges, which affect how they migrate through the gel. In order to accurately separate proteins by molecular weight and not by shape or charge, the secondary structure of the protein is unfolded using the anionic detergent sodium dodecyl sulfate (SDS) and a reducing agent. The SDS molecules form a complex with the protein, negating its inherent charge. The reducing agent breaks covalent bonds that link protein subunits.
After denaturation, the mixture of proteins is added into depressions (or “wells”) within a gel, and then an electrical current is passed through the gel. Because the SDS-protein complex has a strong negative charge, the current drives the proteins through the gel towards the positive electrode. At first glance, a polyacrylamide gel appears to be a solid. On the molecular level, the gel contains channels through which the proteins can pass. Small proteins move through these holes easily, but large proteins have a more difficult time squeez- ing through the tunnels. Because molecules of different sizes travel at different speeds, they separate into discrete “bands” within the gel. After the current is stopped, the bands are visualized using a stain that sticks to proteins.
PROTEIN EXPERIMENTS:
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Proteins and SDS-PAGE
ARTICLES ABOUT PCR:
VIDEOS ABOUT SDS-PAGE
PROTEIN TROUBLESHOOTING GUIDE:
https://www.edvotek.com/site/pdf/Protein_Troubleshoot.pdf
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