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/lab managerbefore 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.
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.
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 AgCl 2. 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.
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 121 deg C 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 <100 deg C
- Open door.
- Remove contents using gloves and immediately tighten all caps.
- To measure the absorbance of a solution in the short-wave range (<300 nM) use the quartz cuvettes. Disposable plastic cuvettes are available for reading in the visible range.
- 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 is OFF 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.
- Press the cell key until cell #1 is in position. Press the Set Reference key to blank against the appropriate buffer. Press the cell key to advance to read the next sample.
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