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Chemical & Physical Safety 1. Handle chemicals and equipment with care. Be aware of locations of showers, eyewashes, fume hoods and first aid assistance. 2. Know what you are handling, several chemicals used in the laboratory are hazardous. Refer to Material Safety Data Sheets (MSDS) supplied by manufacturers or on the Web. Take note especially of the following: Phenol can cause severe burns Acrylamide potential neurotoxin Ethidium Bromide mutagen These and other chemicals are not harmful is used properly: always wear gloves when using potentially hazardous chemicals and never mouth pipette them. 3. If a spill occurs, immediately call the lab instructor or lab staff member. Remove contaminated clothing immediately. Flush skin with water for at least 5 minutes, wash with mild soap and water. Seek medical advice. Contain spilled material in appropriate containers and evaluate the area if necessary. 4. Dispose of all reagents properly. Halogenated, organic, heavy metal, ethidium bromide and solid waste will have proper disposal containers. 5. Never operate equipment you are not familiar with without first consulting the users manual. If the manual is not available, ask the lab instructor or a member of the lab staff for assistance. Be extra careful when plugging in equipment. Always check the voltage requirement; take not is 110- or 220 V. Use a transformer when necessary. Voltage used for electrophoresis is sufficient to cause electrocution. Cover buffer reservoirs during electrophoresis and always turn of the power supply and unplug the power source before removing the gel. 6. General Housekeeping. All common areas should be kept free of clutter and all dirty glassware should be dealt with appropriately. Keep your work areas clean. Since you will use common facilities, all solutions and everything stored in an incubator, refrigerator, etc., must be labeled. In order to limit confusion, each person should use his initials or another unique designation for labeling plates, etc. Cell & Molecular Biology Laboratory: MKCCanlas
Biology Department, School of Science & Engineering Ateneo de Manila University
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Start your data entry on page 5. For each exercise (start new experiments on new pages), an entry is expected in your lab notebook with the following format: 1. 2. 3. 4. 5. 6. 7. Experiment Title: Date/s performed: Outline/Flowchart of Procedure: Reagent Preparations (if necessary): Other Calculations (if necessary): Notes/Reminders (if any): Results:
Items 1 3 are expected to be filled out before class hours. Lab notebooks will be subject to spot-checks during lab period. Write everything that you do in the laboratory in your lab notebook. The notebook should be organized by experiment and should not be organized as a daily log. Buffer compositions (unless standard or included in a kit), biological reagents and special equipment are noted. All results for the experiment are expected to be found in your notebook. These may be in the form of observations, data tables, drawings or photographs (pasted). All should be PROPERLY labeled with the date, identification and source. Lab Reports. A well-researched paper summarizing the each experiment and the results will be submitted. Formal lab reports (group report, unless otherwise stated) must be submitted a week after the exercise is finished. Late lab reports will not be accepted. Automatic zero points for that exercise. Deadlines shall be strictly followed. Points will be deducted for not following directions. Plagiarism is a major offense and subject to sanction or disciplinary action. If a student is absent during the time the experiment is performed, he/she may contribute to the lab report, however, 25% will be deducted from the lab report grade for his/her individual grade. Cell & Molecular Biology Laboratory: MKCCanlas
Biology Department, School of Science & Engineering Ateneo de Manila University
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II.
III.
IV.
V.
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CAUTION: No-nos when using micropipettors 1. Do NOT use without putting a tip on the pipettor; the barrel should never touch the liquid, only the tip. 2. Do NOT lay down the pipettor when its tip contains liquid. Keep it vertical! 3. Do NOT attempt to set the volume above or below the stated range of the device. Pay careful attention to the volume scale and the range! 4. Do NOT let the plunger snap back after taking up or ejecting liquid this damages the piston. Maintain steady control of the plunger speed. Familiarizing yourself with micropipettors 1. Examine your set of micropipettors. We will be using as many as five different pipettors and they should cover the range from 0.2 to 1000 microliters (L). Sometimes the pipettors (and also the accompanying tips) are color-coded by size, clear or white for smallest, yellow for mid-range, blue for largest volume, but this is not always the case. Look at your set for the presence or absence of color-coding on each pipettor. 2. Locate working parts. Pick up any one micropipettor. Identify (but do not manipulate yet) the plunger on the top of the device, the digital volume readout on the body, the separate tip ejector plunger, and the shaft that accepts the tip. 3. Insert a tip onto the shaft of the pipettor. a. Get the correct tip to fit your pipettor. Color coding may help but is not always present (e.g. yellow tip to pipettor with yellow top). b. Press firmly to create an airtight seal between the barrel of the shaft and the tip so the instrument can do its work in drawing up the precise volume of liquid. Never insert a pipettor into liquid without a tip this would ruin the piston that measures the precise volume. 4. Practice operating the plunger on the top of the device as follows: (we are not involving liquid yet) a. Place your thumb on the plunger. Use the rest of your fingers to grip the instrument with your index finger resting in the slot. b. Gently, with steady pressure, depress the plunger till you reach the first stop, that is, till you can detect resistance. Then depress further, till you get to the second stop. (One version of a Cell & Molecular Biology Laboratory: MKCCanlas
Biology Department, School of Science & Engineering Ateneo de Manila University
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Locate the volume adjustor: sometimes this is the plunger itself or sometimes it is a knob near the top of the pipettor. Next, look at the volume readout window; different brands of pipettors have different approaches to the readout scale. Your pipettor may have a direct digital readout with the decimal place clearly indicated for ease of use or it may have a micrometer readout setting that varies with the size of the pipettor. A direct readout is easy, no need to explain, the decimal points are shown. But the micrometer readouts require more attention. See appendix A for detailed directions if you are using one of these pipettor sets; tricky! Do not attempt to set the volume beyond the pipettes minimum or maximum value doing so damages the gears. That is why it is so important to know how to read the range and volume readout of each kind of pipettor. P1000
P100
P20
P10
1 7 5
750 L
1 5 5
15.5 L
0 0
100 L
8 5
8.5 L
Digital display for Gilson Pipetman micropipettors DO NOT DIAL PAST THE LIMITS OF THE PIPETTE! Using micropipettors: Working with Sample Liquid. 1. First arrange your materials on your workspace for easy access, and sit at a height so that you can rest your elbow on the work surface. 2. Pick up a micropipettor with a tip in place. If it is the small-volume pipettor, set the volume to 4 L. If it is the mid-range pipettor, set the volume to 75 L. If the largest volume pipettor, set the volume to 350 L. 3. Obtain a microcentrifuge tube containing colored liquid sample. Hold the opened tube firmly in one hand at nearly eye level to observe the movement of the liquid as you operate the pipettor. Cell & Molecular Biology Laboratory: MKCCanlas
Biology Department, School of Science & Engineering Ateneo de Manila University
P2
1 7 5
1.75 L
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Trial 1 2 3 4 5
1000 L drop
20 L
Accuracy is an expression of how close a measurement instrument comes to the true or accepted value 1.0 mL of water should weigh 1.0 g. Look at your values. Comment on the accuracy of your micropipettor. References: Oklahoma City Community College Biotechnology Program (2009) Learning to Use a Micropipettor. Retrieved 15 June 2009 from http://www.occc.edu/ BBDiscovery/documents/Modules/ learn_micropip.htm Oklahoma City Community College Biotechnology Program (2009) Additional Exercises in Using Micropipettors. Retrieved 15 June 2009 from http://www.occc.edu/BBDiscovery/documents/ Modules/Micropipetting_Exercise.htm SF Base (1994) Skill building Activity 1: Small volumes. Retrieved 15 June 2009 from http://www.usc.edu/org/cosee-west/Jun07Resources/PipetteUsetraining.pdf
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Example: Prepare 500 ml of 0.5 M NaCl (NaCl is 58.55 g/mol). How many grams of NaCl do you need? 0.5 mol/L = X= Xg 58.55 g/mol 14. 6 g NaCl 1 0.5 L
Dissolve 14. 6 g NaCl powder in a final volume of 500 mL water. 3. Preparation of working solutions from concentrated stock solutions Many buffers in molecular biology require the same components (Tris, EDTA, SDS, etc.) but often in varying concentrations. To avoid having to make every buffer from scratch, it is useful to prepare several concentrated stock solutions and dilute as needed.
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EDTA
500 mM X = X =
1 mM 50 mL 0.1 mL
Combine 0.5 mL of 1M Tris with 0.1 mL of 0.5M EDTA with 49.4 mL of water. 4. X solutions Many buffers are prepared as concentrated solutions, example 50X TAE buffer, 10X reaction buffer, and are often diluted so that the final concentration of the buffer in the reaction is 1X (or as specified). Example: Tris-Acetate-EDTA buffer is prepared as a 50X stock solution. Electrophoresis makes use of 1X TAE. Prepare 1L of 1X TAE from the 50X TAE stock solution. Ci x Vi = Cf x Vf Ci Vi Cf Vf = 50X = ? = 1X = 1L 50X Vi = Vi = = 1X 1 L 0.02 L 20.0 mL
Mix 20.0 mL 50X TAE with enough water to bring the final volume of the solution to 1 L. Steps in solution preparation 1. Refer to the laboratory manual for any specific instructions on preparation of the particular solution and the bottle label for any specific precautions in handling the chemical. 2. Weigh out the desired amount of chemical(s). Use an analytical balance if the amount is less than 0.1 g. 3. Pour the chemical(s) in an appropriate size beaker with a magnetic stir bar. 4. Add less than the required amount of water. Prepare all solutions with double-distilled water. When the chemical is dissolved, transfer to a graduated cylinder and add the required amount of distilled water to achieve the final volume. An exception is when preparing solutions containing agar or agarose. Weigh the agar or agarose directly in the final vessel. Cell & Molecular Biology Laboratory: MKCCanlas
Biology Department, School of Science & Engineering Ateneo de Manila University
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7. Autoclave, if possible, at 121C, 15 psi for 20 minutes. Some solutions cannot be autoclaved; for example, SDS. These should be filter-sterilized through a 0.22-m filter. When autoclaving liquids, do NOT tighten the caps to avoid pressure build up. Cover caps completely with aluminium foil. Media for bacterial cultures must be autoclaved the same day it is prepared, preferably within an hour or 2. Store at room temperature and check for contamination prior to use by holding the bottle at eye level and gently swirling it. 8. Solid media for bacterial plates can be prepared in advance, autoclaved, and stored in a bottle. When needed, the agar can be melted in a microwave, any additional components, e.g., antibiotics, can be added, and the plates can then be poured. 9. Concentrated solutions, e.g., 1M Tris-HCl pH = 8.0, 5M NaCl, can be used to make working stocks by adding autoclaved double-distilled water in a sterile vessel to the appropriate amount of the concentrated solution. 10. Glass and plasticware used for molecular biology must be scrupulously clean. Glassware should be rinsed with distilled water and, if needed, autoclaved or baked at 150C for 1 hour. Plasticware, such as pipettes and culture tubes, is often supplied sterile. Tubes made of polypropylene are turbid and resistant to many chemicals, like phenol and chloroform; polycarbonate or polystyrene tubes are clear and not resistant to many chemicals. Micropipette tips and microcentrifuge tubes should be autoclaved before use. Boxes and jars of tips and tubes should be dried overnight in a drying oven after autoclaving prior to use. Reference: Harisha, S. 2007. Biotechnology Procedures and Experiments Handbook. USA/India: Infinity Science Press LLC.
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LB (Luria Bertani) Medium (per liter) Bacto-tryptone 10 g Bacto-yeast extract 5g NaCl 10 g Autoclave. Where indicated add ampicillin to 50g/mL after autoclaved solution has cooled to 50oC. LB plates with ampicillin, (per liter) Add 15 g agar to 1 liter of LB medium. Autoclave. Allow the medium to cool to 55 oC before adding ampicillin (50 g/mL final concentration). Pour 30 - 35 mL of medium into 100 mm petri dishes. Let the agar harden. Store at room temperature (for 1 week) or at 4oC (for 1 month). ANTIBIOTICS Ampicillin Prepare a 50 mg/mL solution of ampicillin in H 2O. Stock solutions of antibiotics dissolved in H 2O should be sterilized by filtration through a 0.22-micron filter. Store solutions in light-tight containers at -20oC. STOCK SOLUTIONS 10% Sodium dodecyl sulfate (SDS) Dissolve 10 g of electrophoresis-grade SDS in 90 mL of H2O. Heat to 68oC to assist dissolution. . Adjust volume to 100 mL. CAUTION: wear a mask when weighing SDS. There is no need to sterilize 10% SDS. 1 M Tris Dissolve 12.1 g Tris base in 80 mL of H2O Adjust the pH to the desired value by adding concentrated HCl: pH 7.4: ~ 7.0 mL pH 8.0: ~ 4.2 mL pH 9.0: ~ 0.8 mL Allow the solution to cool to room temperature before making the final adjustments to the pH. Make up the volume of the solution to 100 mL. Dispense into aliquots and sterilize by autoclaving. *NOTE: if you are using powdered Tris-HCl, check the pH before adjusting with additional HCl. 0.5 M EDTA (pH 8.0) Add 186.1 g of disodium ethylene diamine tetraacetate-2 H2O to 800 mL of H2O Stir vigorously on a magnetic stirrer Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. 2M Glucose Dissolve 18 g L-glucose (Dextrose) in 45 mL of H2O. Adjust volume to 100 mL. Filter sterilize. 10N NaOH Cell & Molecular Biology Laboratory: MKCCanlas
Biology Department, School of Science & Engineering Ateneo de Manila University
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Gel loading buffer, Type III 6X buffer: 0.25% bromophenol blue 0.25% xylene cyanol 30% glycerol in H2O. Store at 4oC. Reference: Sambrook, J, EF Fritsch and T Maniatis. 1989. Molecular Cloning: A Laboratory Manual. 2nd Edition. Cold Spring Harbor Laboratory Press NY.
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