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Antibiotics should be added to culture media after autoclaving at temperatures lower than 60°C.
E.coli Growth Curve:
The growth of E. coli can be divided into distinct phases.
They are as follows:
1. ‘Lag phase’ occurs after the dilution of the starter culture into the fresh medium. Cell division is slow as the bacteria adapt to the fresh medium.
2. After 4-5 hours the culture enters ‘logarithmic (log) phase’, where bacteria grow exponentially.
3. Cells enter ‘stationary phase’ (approximately 16 hours) when the available nutrients are used up. The cell density remains constant in this phase.
4. Eventually the culture enters the ‘phase of decline’, where cells start to lyse, the number of viable bacteria falls, and DNA becomes partly degraded.
5. The growth curve of a bacterial culture can be measured colorimetrically by reading the OD at 600 nm.
Storage of Bacterial Strains in Stab Agar:
Most E. coli strains can be stored for one or two years in stab agar.
1. Use airtight, autoclaved vials containing approximately 1 ml of medium.
2. Inoculate each vial with a single colony.
3. Leave the cap of the vial slightly loose and incubate overnight at 37°C.
4. Seal the vial tightly and store at room temperature in a dark place.
5. Streak onto LB plate containing appropriate antibiotics for a single colony to revive a stored strain.
Storage of Bacterial Strains in Glycerol Solution:
Most strains of bacteria including E. coli, and fungi (spores are preferred) can be stored for one or two years in glycerol at -20°C. At -70°C they can be stored almost for lifetime.
1. Add 0.15 ml of glycerol (100%) solution in 2.0 ml screw-cap vials.
2. Autoclave for 15 minutes at 121°C. Cool and store them at room temperature.
3. Transfer one drop (0.85 ml) of an overnight-grown logarithmic-phase culture into a vial.
4. Vortex, store at -20 or -70°C (-70°C storage is preferred for long-term maintenance).
5. To revive a stored strain, streak onto LB plate containing appropriate antibiotics for a single colony.
Decontamination of Ethidium Bromide (EtBr) Solutions:
EtBr is a powerful mutagen and is moderately toxic. Wear gloves when working with EtBr.
When EtBr concentration is >0.5 µg/ml
1. Add sufficient water to reduce the concentration of EtBr to < 0.5 µg/ml.
2. Add 1 volume of 0.5 M KMnO4. Mix and add 1 volume of 2.5 N HCl (210 ml HCl in 1 liter water). Mix and allow the solution to stand for several hours at room temperature.
3. Add 1 volume of 2.5 N NaOH (100 g NaOH in 1 liter water). Mix, and discard the supernatant solution.
When EtBr concentration is 0.5 µg/ml
(Electrophoresis buffer contains 0.5 µg/ml EtBr)
1. Add 29 g Amberlite XAD-16 (Rohm and Hass) for 1 liter of solution.
2. Store the solution for 12 hours at room temperature, shaking it intermittently.
3. Filter the solution through a Whatman No. 1 filter paper and discard the filtrate.
4. Seal the filter paper and Amberlite resin in a plastic bag, and dispose of the bag in the hazardous waste.
1. Molar Solutions:
A molar solution is one in which 1 liter of solution contains the number of grams equal to its molecular weight.
Example – To make up 100 ml of a 5 M NaCl solution
58.456 (mw of NaCl) g x 5 moles x 0.1 liter = 29.29 g in 100 ml solution
Percentage (w/v) = weight (g) in 100 ml of solution
Percentage (v/v) = volume (ml) in 100 ml of solution.
Example – To make a 0.7% solution of agarose in TBE buffer, dissolve 0.7 g of agarose and bring up volume to 100 ml with TBE buffer.
Many enzyme buffers are prepared as concentrated solutions, for example as 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.
Example – 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.
4. Preparation of Working Solutions from Concentrated Stock Solutions:
Many buffers in molecular biology require the same components 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.
Example – To make 100 ml of working TE buffer (10 mM Tris and 1 mM EDTA), combine 1 ml of a 1 M Tris solution and 0.2 ml of 0.5 M EDTA stock and 98.8 ml sterile water.
The following formula is useful for calculating the amounts of stock solution needed –
Ci x Vi = Cf x Vf
Where,
Ci = initial concentration, or concentration of stock solution;
Vi = initial volume or amount of stock solution needed
Cf = final concentration, or concentration of desired solution
Vf = final volume or amount of desired solution.
Steps in Solution Preparation:
1. Weigh out the desired amount of chemical(s). Use an analytical balance if the amount is less than 100 mg.
2. Place chemical(s) in appropriate size beaker with a stir bar.
3. Add less than the required amount of water. Prepare all solutions with double distilled water (in carboy).
4. 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 volume of solution.
5. If the solution needs to be at a specific pH, check the pH of fresh buffer solutions and follow instructions for using a pH meter.
6. Autoclave, if possible, at 121°C for 20 minutes. Some solutions cannot be autoclaved, for example, SDS. These should be filter sterilized through a 0.22 µm filter. Media for bacterial cultures must be autoclaved on the same day of preparation, 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.
7. 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, such as antibiotics, can be added and the plates can then be prepared.
8. Concentrated solutions, example 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.
Glass-and plasticware used for molecular biology must be thoroughly 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 150°C for 1 hour. For experiments with RNA, glassware and solutions are treated with diethyl-pyrocar- bonate (DEPC) to inhibit RNases which can be resistant to autoclaving.
Plasticware such as pipettes and culture tubes are often supplied sterile. Tubes made of polypropylene are resistant to many chemicals, like phenol and chloroform; polycarbonate or polystyrene tubes are clear but not resistant to many chemicals.
Make sure that the tubes you are using are resistant to the chemicals used in your experiment. Micropipette tips and microfuge tubes should be autoclaved before use.
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, such as 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.
Autoclave Operating Procedures:
Place all materials to be autoclaved in an autoclavable tray. All items should have indicator tape. Separate liquids from solids and autoclave separately. Make sure lids on all bottles are loose. Do not crowd large numbers of items in tray. For all items to reach the appropriate temperature, one must allow sufficient air/steam circulation.
1. Make sure chamber pressure is at 0 before opening the autoclave.
2. Place items to be autoclaved in the autoclave and close it.
3. Temperature would be preset at 121°C, but double-check and change if necessary.
4. Autoclave typically for 20 minutes.
At the end of the cycle, check that –
1. The chamber pressure is at 0
2. The temperature is <100°C
Open autoclave remove contents using gloves and immediately tighten all caps.
Chemicals:
A number of chemicals used in the molecular biology 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 the chemicals.
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 hazardous:
a. Phenol causes severe burns
b. Acrylamide is a potential neurotoxin
c. Ethidium bromide is carcinogenic
These chemicals are not harmful if used properly. Always wear gloves when using potentially hazardous chemicals and never mouth-pipette 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.
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 minutes to 24 hours after exposure. Therefore, always wear appropriate eye protection when using UV lamps.