A collection of ImmuniBee's laboratory protocols. Feel free to use and adapt them as you choose!
A collection of ImmuniBee's laboratory protocols. Feel free to use and adapt them as you choose!
LB agar plates are commonly utilized for the growth of E. coli and other bacteria in research, providing nutrient-rich media. Additionally, antibiotics are often added to LB agar plates for the purposes of bacterial selection in cloning experiments. Agar content typically varies from 1% to 2%, although this protocol yields 1.2% agar plates.
Source: 2022 UBC-Okanagan
Note: If you are pre-making plates for later use, ensure they are completely cool and store at 4°C upsidedown in a plate bag, parafilming the plates is optional but recommended.
Sequence verification of plasmids by digestion with restriction endonucleases, followed by agarose gel electrophoresis to visualize digested DNA. The restriction enzymes, DNA, water, and buffer are added to a PCR tube, incubated at 37 °C for the appropriate time, and heat inactivated at 65 °C for 20 minutes. Incubation and heat inactivation can be performed in a thermal cycler, or in heating mantles and water baths. Gel electrophoresis visualization of analytical digestions can be simulated through programs such as Benchling or SnapGene, and results can be compared with simulations to verify plasmid sequences.
Note: If the plasmid DNA concentration is known use 1μg, if the concentration is unknown use 5μL.
Antibiotic stocks at a concentration of 1000X are a convenient way to have the necessary antibiotic on hand and are easily accessible for aliquoting antibiotics for selection agar plates, liquid selection media and other solutions.
Source: 2022 UBC-Okanagan
Note: Aliquoting antibiotic solution into small working stocks prevents the need to thaw too much antibiotic solution at once, which is highly recommended when working with light-sensitive or heat-sensitive antibiotics.
Antibiotic Concentration
A B C D
Cryostocking is an excellent way to preserve a culture in a way that is suitable for long-term storage. The addition of glycerol prevents the formation of ice crystals in liquid media, keeping cells viable once frozen. However, exposing cells to glycerol at temperatures above freezing is increasingly harmful with time, thus cryostocks must be placed on dry ice immediately after glycerol has been added. For the same reason, the number of times an individual cryostock is thawed and refrozen should be minimized to ensure the long-term viability of cells.
source: UBC-Okanagan 2022
The small-scale, rapid extraction and purification of plasmid DNA from E. coli cultures grown overnight in LB media, utilizing NEB’s Monarch Plasmid DNA Miniprep Kit. This protocol is designed by NEB for use with their Monarch Plasmid DNA Miniprep Kit. Miniprep plasmid extractions are generally suitable for a yield of 5 to 50 μg of plasmid DNA. Plasmid extraction is achieved through the pelleting of overnight cultures, followed by resuspension, lysis and DNA denaturation, neutralization of lysis buffer, transfer to a spin column, washing away of impurities, and elution of DNA.
Source: 2022 UBC-Okanagan
Note: Be careful not to shear chromosomal DNA by vortexing or vigorous shaking. Firmly inverting the tube promotes good mixing, important for full neutralization.
Note: Spin time should not be less than 2 minutes. Careful handling of the tube will ensure no debris is transferred and the 2 minute recommended spin can be successfully employed to save valuable time. For culture volumes > 1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Also, longer spin times will result in a more compact pellet that lowers the risk of clogging the column.
This protocol was adapted to fit the equipment and materials available in the lab.
Gel electrophoresis is a helpful protocol to distinguish DNA by its length in base pairs. It can either be used for further purification or solely visualization. The theory behind Electrophoresis is using an electrical field propagated by two oppositely charged electrodes to mode the negatively charged DNA through the gel matrix (made out of agarose) toward the positive electrode. This is helpful since longer DNA fragments tend to move slower through the gel than shorter fragments: enabling the user to determine the approximate length of the DNA by comparing it to a DNA ladder with standard known base pair lengths.
Original Source: Agarose Gel Electrophoresis
Day 1:
Note: clean weighting boat with RO water immediately after using
*Tip* Place inversely a 25 or 50 ml volumetric flask in the top of the Erlenmeyer Flask to hear when boiling gas is being produced.
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00:05:00
Note: This chemical binds to DNA : 1µL of CyberSafe per 50 ml of gel.
Conversely: (Optional) Add ethidium bromide (EtBr) to a final concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light.
CRITICAL CAUTION: EtBr is a known mutagen. Wear a lab coat, eye protection and gloves when working with this chemical.
The orange rubber bands need to be placed correctly to prevent leakage
Note: immediately clean the Erlenmeyer Flask with RO water after using
Note:: make sure the cybersafe does NOT get stuck on the corners by using a pipette.
Note: if the gel is not to be used immediately place a paper towel on top to prevent degardation and avoid particles from falling in.
00:25:00
Note: Avoid bubbles
*Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated; 2) it contains a high percentage of glycerol that increases the density of your DNA sample causing it to settle to the bottom of the gel well, instead of diffusing in the buffer.
Note: When loading the sample in the well, maintain positive pressure on the sample to prevent bubbles or buffers from entering the tip. Place the very top of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample out and watch as the sample fills the well. After all of the samples are unloaded, push the pipettor to the second stop and carefully raise the pipette straight out of the buffer.
Note: Black is negative, red is positive. The DNA is negatively charged and will run towards the positive electrode. Always Run to Red.
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*Tip* If you will be purifying the DNA for later use, use long-wavelength UV and expose it for as little time as possible to minimize damage to the DNA.
CRITICAL When using UV light, protect your skin by wearing safety goggles or a face shield, gloves and a lab coat.
The seamless assembly of one or more DNA sequences into a plasmid backbone through the use of Type IIS restriction enzymes and DNA ligase. Type IIS restriction endonucleases are advantageous due to the fact that they cut outside of their recognized restriction sites. This allows for the custom design of sticky ends for one-pot, one-step assemblies with multiple inserts.
Source: 2022 UBC-Okanagan
Note: calculate for equimolar concentrations based on concentration of backbone and insert DNA, using 100 ng of backbone DNA.
Thermal Cycler Conditions
Temp and Duration Concentration
PCR is a DNA amplification technique that is commonly used in synthetic biology.
Resuspension of template
PCR
Table1
| A | B | |
|---|---|---|
| 1 | Reagent | Volume for 1 Rxn (uL) |
| 2 | Nuclease-Free Water | 8 |
| 3 | PCR Mastermix | 12.5 |
| 4 | Resuspended template | 2 |
| 5 | 10uM forward primer | 1.25 |
| 6 | 10uM reverse primer | 1.25 |
| 7 | Total | 25 |
Table2
| A | B | C | D | |
|---|---|---|---|---|
| 1 | Step | Temperature (C) | Time | # of Cycles |
| 2 | Initial Denaturation (1 cycle) | 94 | 30s | 1 |
| 3 | Denaturation | 94 | 30s | 30 |
| 4 | Primer Annealing | *55 | 30s | |
| 5 | Extension | 72 | 1 min/kb | |
| 6 | Final Extension (1 cycle) | 72 | 5min | 1 |
| 7 | Hold | 4 | - | - |
*Annealing temperatures vary by primer and are individually calculated depending on the DNA sequence and length.
SDS-PAGE (or sodium dodecyl sulphate–polyacrylamide gel electrophoresis) is a technique for the separation of proteins based on size. Negatively charged SDS molecules bind to proteins, providing a consistent mass-to-charge ratio to all proteins. These samples are loaded into a polyacrylamide gel with an electrophoresis buffer, and subjected to a voltage that causes variable migration of the proteins through the gel based on size due to the SDS negative charge. The mesh of the gel allows for faster travel of smaller proteins, resulting in separation. Separated protein bands are then visualized through staining.
Source: 2022 UBC-Okanagan
i. Weigh 100mg of Bromophenol blue, 0.747g of Tris Base, 2g of Sodium dodecyl sulphate, and place into a volumetric flask.
ii. Fill a volumetric flask halfway and dissolve contents with H2O.
iii. Add 5mL of beta-mercaptoethanol and top up to the 50mL line of the flask.
Note: we made a 300mL solution. So by the 50:10:40 ratio, the solution comprised 150mL MeOH, 30mL Acetic Acid, and 120mL H 2O.
Method for viewing live bacteria in an optical microscope. It permits the examination of the living microorganisms, which involves fixation of microbial suspension into a drop of fluid over the glass slide. Therefore hanging drop method is the process, in which the microorganisms are suspended into a drop of fluid. To verify if your cells are present refer to the notes below:
Sources: Microscope Master, Biology Reader
Aseptic liquid inoculation technique.
Test if the loop has cooled enough by pressing the loop against a clear area of the agar plate. If the agar melts, cool the loop for another 5-10 seconds.
Optical density (turbidity) is a standard measurement method for determining cell growth in liquid media utilizing a spectrophotometer. The absorbance given by the spectrophotometer can be used to determine the density and growth of bacteria in a liquid media by measuring the amount of light that passes through the culture.
Source: 2022 UBC-Okanagan
Note: This step should be done prior to each reading for maximum accuracy. Note: The spectrophotmeter should read the same negative number each time.
A spread plate inoculation is a type of inoculation where the bacteria is used to inoculation the entirety of the plate in an even motion.
A streak plate inoculation is used to inoculate a plate in 4 quadrants, allowing for the easy visulaization of individual colonies.
Test if the loop has cooled enough by pressing the loop against a clear area of the agar plate. If the agar melts, cool the loop for another 5-10 seconds.
Bacillus subtilis strains are supplied as frozen cultures and shipped on dry ice. Store the stock at -80C. For propagation, remove the tube from the freezer, and scratch off some from the surface of the frozen stock using a sterile loop. Streak onto a LB plate, seal the plate with Parafilm and incubate at 37C overnight. Bacillus plates can be stored at 4C for 1 month. Use fresh bacteria for transformation.
B. subtilis can be grown at 37C in 2xYT medium. Under optimal conditions doubling time is 30 min.
PBS (Phosphate Buffered Saline)
To make 1L of PBS
| 10x | 1x | |
|---|---|---|
| NaCl | 80g | 8g |
| KCl | 2g | 0.2g |
| Na2HPO4 | 14.4g | 1.44g |
| KH2PO4 | 2.45g | 0.245g |
10% Formalin Solution
Bee Dissection and Tissue Imaging Protocol
Day 1:
Day 2:
Preparation of bees for dissection, dissection methods, and imaging preparation.
In a well plate, remove paraformaldehyde, fill with fresh PBS then repeat this 2 more times.
PBS with (Triton-X100); [0.1% of the final volume], snip the tip of a pipette tip because it is so thick and viscous. Helps to make the cell membranes more permeable to the dyes.
Link to source paper. This buffer is a material needed in order to bulk extract the RNA of whole bees using acid-phenol method
Day 1:
Following safety procedures (http://www.sciencelab.com/msds.php?msdsId=9927539)