Appendix I -- Operation of the Pipettman16:
The Pipettman, an adjustable digital variable volume microliter pipettor, is to be used only for dispensing the enzyme and solutions in this experiment. The volume indicators consist of three numbered dials and are read from top to bottom. Carefully read the volume of the pipettor you are using since the pipettors we have in the lab are available in several standard volumes from 20 µl to 1000 µl. The digits represent milliliters or microliters depending on which model unit you are using.
- To set the volume, hold the Pipettman in one hand and turn the volume adjustment knob with the other hand until the correct volume shows on the digital indicator. If you pass the desired setting, turn the dial a few digits above the desired setting and reset the volume. The lab pipettors are remarkably free of mechanical backlash, however, it is considered good practice to consistently overset the actual volume required and ensures that the best accuracy is obtained.
- Attach a new disposable tip to the pipet shaft. Press firmly to ensure a positive airtight seal using a slight screwing action. NEVER USE THE PIPETTOR WITHOUT A TIP ATTACHED.
- Depress the plunger to the FIRST STOP. This part of the stroke is the calibrated volume displayed on the digital volume indicator.
- Holding the pipettman vertically, immerse the disposable tip into the sample liquid to the proper immersion depth generally 3-5 mm into the sample being tested.
- Allow the pushbutton to return SLOWLY to the up position. NEVER LET IT SNAP UP!!
- Wait a few seconds to ensure that the full volume of sample is drawn into the tip.
- Withdraw the tip from the sample liquid slowly. Should any liquid remain on the outside of the tip, wipe it carefully with a lint-free tissue, taking care not to touch the tip orifice. DO NOT WIPE THE TIP WITH A TISSUE.
- To dispense samples, touch the tip end against the sidewall of the receiving vessel about midway down, not at the very bottom, and depress the plunger slowly to the FIRST STOP. Wait 2-3 seconds. Then press the plunger to the SECOND STOP (bottom of stroke), expelling any residual liquid in the tip.
- With the plunger fully depressed, withdraw the Pipettman from the vessel carefully, with the tip sliding along the wall of the vessel.
- Allow the plunger to return to the UP position.
- Discard the tip by depressing the tip ejector button most of the pipettors we have in the undergraduate lab do not have ejector buttons so you can pull the tip off with gloves and dispose into the disposable container designated by the TA. A fresh tip should be used for each sample to prevent sample carryover.
Appendix II -- UV-Vis Operation
Guidelines for measuring a UV-Vis spectrum using the Perkin Elmer Lambda 35 UV/VIS Spectrometer for the Protein Assay Experiments with Automatic Cell Feeder
- Turn on computer
- Insert your personal jump drive into the top USB port on the front of computer
- On the main screen, double click on the Perkin Elmer UV Win Lab icon on the top right corner of the screen
- At Perkin Elmer login message, User name must say student or students click OK. The Base Methods appears in the left-hand part of the screen
- Left click on Protein Assay Method Module. Sample information window then loads. Under sample ID, identify the samples; under concentration, note the concentrations in micrograms/mL. Samples box at top of the screen should read 0. Do not change the cell or carousel numbers. Press Start button at top of page. Machine scans an air blank. At this point, there should be nothing inside the cell changer.
- Message appears to instruct to remove sample. Open sample door and place your blank in position 1 (extreme lower left port on cell changer), which is scanned as a blank to produce a spectral background. When handling a cuvette, never touch the clear sides of the cuvette since it is always in the path of the light beam. The arrow on the side of the cuvette should be placed in the light beam. Keep the exterior of the cuvette clean and dry before inserting it. Close the sample door and click on OK.
- After about a minute, another message appears asking you to insert your standards into cells 2 thru 8, respectively, depending the number of standards (you can run up to seven standards/samples simultaneously). Open the sample door, remove the blank, and insert the standards in correct order. Close the door and click on OK. Machine begins to scan standards and overlay the graphs one at a time. Once the standards have been run, it will ask you to load the final samples follow the instructions. Take out your standards and insert the five samples into positions 2 to 6. Then click on OK.
- Message then appears indicating that all samples in the table have been run; click on OK. Calibration message appears, click on OK.
- Now on the left side of screen, click on the Output, then click on File on the top, and select Report. Click on OK to Print and it will print your graph and or your list of absorbance v. concentration values.
- Now save the data to your jump drive that you inserted previously. Go to File, select Save Results, and then choose As a New Task. You then name your file and save task message. The computer creates a new folder by that name on your removable jump drive
To safely remove the jump drive from the computer, look for the green icon on the lower bottom right side called Safely Remove Hardware Icon. Click it and a Safely remove hardware message opens. Select the task and click Stop a hardware device window opens. Select your device from the menu and click OK. A message appears at bottom of screen indicating that it is safe to remove Hardware. Now, pull out the jump drive or USB device.
16 Adapted from: VWR brand Pipettor Instruction Manual, VWR Scientific Products.