A. Chemicals
A number of chemicals used in this laboratory are hazardous. All manufacturers of hazardous materials are required by law to supply the user with pertinent information on any hazards associated with their chemicals. This information is supplied in the form of Material Safety Data Sheets or MSDS. This information contains the chemical name, CAS#, health hazard data, including first aid treatment, physical data, fire and explosion hazard data, reactivity data, spill or leak procedures, and any special precautions needed when handling this chemical. A file containing MSDS information on the hazardous substances used in the Molecular Biology course is kept in the lab. In addition, MSDS information can be accessed on World Wide Web on the Biological Sciences Home Page. You are strongly urged to make use of this information prior to using a new chemical and certainly in the case of any accidental exposure or spill. The instructor must be notified immediately in the case of an accident involving any potentially hazardous reagents.
The following chemicals are particularly noteworthy:
Phenol - can cause severe burns
Acrylamide - potential neurotoxin
Ethidium bromide - carcinogen
These chemicals are not harmful if used properly: always wear gloves when using potentially hazardous chemicals and never mouth-pipet them. If you accidentally splash any of these chemicals on your skin, immediately rinse the area thoroughly with water and inform the instructor. Discard the waste in appropriate containers.
B. Ultraviolet Light
Exposure to ultraviolet light can cause acute eye irritation. Since the retina cannot detect UV light, you can have serious eye damage and not realize it until 30 min to 24 hours after exposure. Therefore, always wear appropriate eye protection when using UV lamps.
C. Electricity
The voltages used for electrophoresis are sufficient to cause electrocution. Cover the buffer reservoirs during electrophoresis. Always turn off the power supply and unplug the leads before removing a gel.
D. General Housekeeping
All common areas should be kept free of clutter and all dirty dishes, electrophoresis equipment, etc should be dealt with appropriately. Since you have only a limited amount of space to call your own, it is to your advantage to keep your own area 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 other unique designation for labeling plates, etc. Unlabeled material found in the refrigerators, incubators, or freezers may be destroyed. Always mark the backs of the plates with your initials, the date, and relevant experimental data, e.g. strain numbers.
II. PREPARATION OF SOLUTIONS
A. Calculation of Molar, % and "X" Solutions.
1. A molar solution is one in which 1 liter of solution contains the number of grams equal to its molecular weight. Ex. To make up 100 ml of a 5M NaCl solution =
58.456 (mw of NaCl) g x 5 moles x 0.1 liter = 29.29 g in 100 ml sol mole liter
2. Percent solutions.
Percentage (v/v) = volume (ml) in 100 ml of solution.
Ex. To make a 0.7% solution of agarose in TBE buffer, weight 0.7 of agarose and bring up volume to 100 ml with TBE buffer.
3. "X" Solutions.
Many enzyme buffers are prepared as concentrated solutions, e.g. 5X or 10X (five or ten times the concentration of the working solution) and are then diluted such that the final concentration of the buffer in the reaction is 1X.
Ex. To set up a restriction digestion in 25 μl, one would add 2.5 μl of a 10X buffer, the other reaction components, and water to a final volume of 25 μl.
B. Preparation of Working Solutions from Concentrated Stock Solutions.
Ex. To make 100 ml of TE buffer (10 mM Tris, 1 mM EDTA), combine 1 ml of a 1 M Tris solution and 0.2 ml of 0.5 M EDTA and 98.8 ml sterile water. The following is useful for calculating amounts of stock solution needed:
Ci x Vi = Cf x Vf , where Ci = initial concentration, or conc of stock solution;
Vi = initial vol, or amount of stock solution needed
Cf = final concentration, or conc of desired solution;
Vf = final vol, or volume of desired solution
C. Steps in Solution Preparation:
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.
Weigh out the desired amount of chemical(s). Use an analytical balance if the amount is less than 0.1 g.
Place chemical(s) into appropriate size beaker with a stir bar.
Add less than the required amount of water. Prepare all solutions with double distilled water (in carboy)
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 in preparing solutions containing agar or agarose. Weigh the agar or agarose directly into the final vessel.
If the solution needs to be at a specific pH, check the pH meter with fresh buffer solutions and follow instructions for using a pH meter.
Autoclave, if possible, at 121EC for 20 min. Some solutions cannot be autoclaved, for example, SDS. These should be filter sterilized through a 0.22μm filter. Media for bacterial cultures must be autoclaved the same day it is prepared, preferably within an hour or two. Store at room temperature and check for contamination prior to use by holding the bottle at eye level and gently swirling it
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.
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.
D. Glassware and Plastic Ware.
Glass and plastic ware used for molecular biology must be scrupulously clean. Dirty test tubes, bacterial contamination and traces of detergent can inhibit reactions or degrade nucleic acid.
Glassware should be rinsed with distilled water and autoclaved or baked at 150EC for 1 hour. For experiments with RNA, glassware and solutions are treated with diethyl-pyrocarbonate to inhibit RNases which can be resistant to autoclaving.
Plastic ware such as pipets and culture tubes are often supplied sterile. Tubes made of polypropylene are turbid and are resistant to many chemicals, like phenol and chloroform; polycarbonate or polystyrene tubes are clear and not resistant to many chemicals. Make sure that the tubes you are using are resistant to the chemicals used in your experiment. Micropipet tips and microfuge tubes should be autoclaved before use.
III. Disposal of Buffers and Chemicals
Any uncontaminated, solidified agar or agarose should be discarded in the trash, not in the sink, and the bottles rinsed well.
Any media that becomes contaminated should be promptly autoclaved before discarding it. Petri dishes and other biological waste should be discarded in Biohazard containers which will be autoclaved prior to disposal.
Organic reagents, e.g. phenol, should be used in a fume hood and all organic waste should be disposed of in a labeled container, not in the trash or the sink.
Ethidium bromide is a mutagenic substance that should be treated before disposal and should be handled only with gloves. Ethidium bromide should be disposed of in a labeled container.
Dirty glassware should be rinsed, all traces of agar or other substance that will not come clean in a dishwasher should be removed, all labels should be removed (if possible), and the glassware should be placed in the dirty dish bin. Bottle caps, stir bars and spatulas should not be placed in the bins but should be washed with hot soapy water, rinsed well with hot water, and rinsed three times with distilled water.
IV. Equipment
A. General Comments
It is to everyone's advantage to keep the equipment in good working condition. As a rule of thumb, don't use anything unless you have been instructed in the proper use. This is true not only for equipment in the lab but also departmental equipment. Report any malfunction immediately. Rinse out all centrifuge rotors after use and in particular if anything spills. Please do not waste supplies - use only what you need. If the supply is running low, please notify either the instructor or the TAbefore the supply is completely exhausted. Occasionally, it is necessary to borrow a reagent or a piece of equipment from another lab. Except in an emergency, notify the instructor.
B. Micropipettors.
Most of the experiments you will conduct in this laboratory will depend on your ability to accurately measure volumes of solutions using micropipettors. The accuracy of your pipetting can only be as accurate as your pipettor and several steps should be taken to insure that your pipettes are accurate and are maintained in good working order. Each pair of students will be assigned a set of pipettors and upon receipt, they should be labeled with the students' name. They should then be checked for accuracy following the instructions given by the instructor. If they need to be recalibrated, do so. We have two different types of pipettors, Rainin pipetmen and Oxford benchmates. Since the pipettors will use different pipet tips, make sure that the pipet tip you are using is designed for your pipettor.DO NOT DROP IT ON THE FLOOR. If you suspect that something is wrong with your pipettor, first check the calibration to see if your suspicions were correct, then notify the instructor.
C. Using a pH Meter.
Biological functions are very sensitive to changes in pH and hence, buffers are used to stabilize the pH. A pH meter is an instrument that measures the potential difference between a reference electrode and a glass electrode, often combined into one combination electrode. The reference electrode is often AgCl2. An accurate pH reading depends on standardization, the degree of static charge, and the temperature of the solution.
Operation of Orion PerpHecT pH Meter
Expose hole on side of electrode by sliding the collar down. Make sure there is sufficient electrode filling solution in the electrode (it should be up to the hole). If not, fill with ROSS filling solution only(Do not use any filling solution containing silver (Ag).
Ensure that sample to be pHed is at room temperature and is stirring gently on the stir plate.
Calibrate the pH meter with the two solutions that bracket the target pH - 4 and 7 or 7 and 10 as follows:
Press the CAL key to initialize the calibration sequence. The last calibration range will be displayed (e.g. 7-4). Press YES to accept or use the scroll keys to select a different range. Press YES to accept.
The number 7 will light up on the left hand side of the screen indicating that the meter is ready to accept the pH 7 standard buffer. Rinse off electrode and place in fresh pH 7 standard buffer solution. The READY light will come on when the value has stabilized. Press YES to accept the value.
The number 4 (or 10) will light up next indicating that the meter is ready to accept the pH 4 (or 10) standard buffer solution. Rinse off electrode and place in fresh pH 4 standard buffer solution. The READY light will come on when the value has stabilized. Press YES to accept the value.
SLP will be displayed. The meter will then go MEASURE mode.
Rinse electrode and place into sample. The READY light is displayed when signal is stable.
D. Autoclave Operating Procedures
Place all material to be autoclaved in a autoclavable tray. All items should have indicator tape. Separate liquids from solids and autoclave separately. Make sure lids on all bottle are loose. Do not crowd large number of items in tray- in order for all items to reach the appropriate temperature, one must allow sufficient air/steam circulation.
Make sure chamber pressure is at 0 before opening the door.
Place items to be autoclaved in the autoclave and close the door. Some
autoclaves require that you also lock the door after it's closed.
Set time - typically 20 minutes.
Temperature should be set at 121EC already, but double-check and change if necessary.
Set cycle: If liquid, set "liquid cycle" or "slow exhaust". If dry, set "dry cycle" or "fast exhaust" + dry time.
Start the cycle. On some autoclaves, the cycle starts automatically at step 5. On others, turn to "sterilize".
At the end of the cycle, check that:
a. the chamber pressure is at 0
b. the temp is <100EC
Open door. (On 3rd floor autoclave, don't push end cycle)
Remove contents using gloves and immediately tighten all caps.
E. Operating Instructions for Spectrophotometer - Pharmacia Ultraspec
 | To measure the absorbance of a solution in the short-wave range (<300 nM) use the quartz cuvettes. |
| Turn the spectrophotometer on - the switch is on the right in the back. |
| Allow the instrument to calibrate. Do not open the chamber during this time. The deuterium lamp isOFF by default. To read absorbance in the UV range, turn the deuterium lamp on as follows after the machine has completed its calibration: Depress the function key until Fn5 is displayed. Press the mode key until d2on is displayed. Press enter. For best accuracy, the deuterium lamp should be warmed up for 20 minutes. |
| Press the function key until Fn0 is displayed. Press enter. Using the up or down arrow keys, enter in the desired wavelength. |
| Prepare a reference cuvette containing the same diluent as your sample. Prepare your sample. |
| Place the reference cuvette in cell #1 and place your samples in cells #2-6. |
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