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TECHNIQUES IN MOLECULAR BIOLOGY – BASIC TECHNIQUES: PIPETING AND STERILE CULTURE Mastery of micropipetting and sterile technique is essential for reliable success in molecular biology. Take these exercises and our discussions about pipetting seriously. Micropipettors A micropipettor* is essentially a precision pump fitted with a disposable tip. The volume of air space in the barrel is adjusted by screwing the plunger in or out of the piston, and the volume is displayed on a digital readout. Depressing the plunger displaces the specified volume of air from the piston; releasing the plunger creates a vacuum which draws an equal volume of fluid into the tip. The fluid in the tip is expelled by depressing the plunger again, but to a further, second 'blow-out' stop. * The nickname is originally derived from a different pipettor brand ('Pipetman'), but still widely used by molecular molecular biologists for all micropipettors. Take the following precautions when using the pipettor: • Never rotate the volume adjustor beyond the upper or lower range indicated on the pipettor. • Never invert or lay down a pipettor with a filled tip (fluid can run into the barrel). • Never let the plunger snap back after withdrawing or expelling fluid (could damage the piston or cause fluid to enter the barrel, and contact the piston – aka ‘fountaining’). • Never reuse a tip that has been used to measure a different reagent, or that has entered a tube containing a different reagent. When in doubt, use a fresh tip. Pipet-Lite (LTS) Pipettors: Pipet-Lite (LTS) is a brand of micro-pipettor used in the laboratory. Pipet-Lite is an air-displacement pipette which uses a magnetic assist and is slightly different than the Gilson pipets used in other labs. Become familiar with this pipet and it’s components. Setting Volume: • Turn the volume lock counter-clockwise to unlock the pipet. The position shown at left below so the volume setting mechanism is unlocked and free to turn. • With the mechanism unlocked, then rotate the plunger button to change volume – counter-clockwise to increase, and clockwise to decrease volume. • When setting the desired volume, first turn the knob slowly clockwise until the desired volume is displayed. Always dial down to the desired volume. • Lock the pipet by turning the volume lock clockwise (see figure for locked position) 1 TECHNIQUES IN MOLECULAR BIOLOGY – BASIC TECHNIQUES: PIPETING AND STERILE CULTURE Operation: • Press the plunger button tot the first stop, and hold it in this position. The magnetic latch will help you sense and hold this position. • Holding the pipet vertically, place the tip into the sample at the proper depth (see tip emersion chart) and relax your thumb pressure on the plunger. Do not let go of the plunger button, or the piston may snap up quickly, resulting in inaccurate measurement. • Pause briefly to ensure the full volume of sample is drawn into the tip. • Withdraw the tip from the sample. If any liquid remains on the outside surface of the tip, wipe it carefully with a lint-free tissue, taking care NOT to touch the tip orifice. • Touch the tip end against the side wall of the receiving vessel and press the plunger slowly, past the first stop. Wait one to two seconds. • Still holding the plunger down, with draw the tip. Then release the plunger. Watch: (http://www.mt.com/us/en/home/supportive_content/specific_overviews/product-organizations/pipe/rainin- pipette-technique-videos.html) Lab Experiment 1 Measurements, Micropipetting, and Sterile Technique Part1 Micropipetting Reagents Tubes of colored Solutions I, II, III & IV Sterile LB broth I. Practice with a small-volume pipettor This exercise simulates setting up a set of typical small molecular biology reactions such as a small restriction enzyme digest, using a 10 µl pipettor. 1. Label three 1.5 ml microtubes on the lid with a permanent marker: A, B & C. 2. Use the matrix below as a checklist while adding solutions to each reaction tube. Use the techniques discussed above for pipetting. Tube Soln. I Soln. II Soln. III Soln. IV A 4 µl 5 µl 1 µl – B 4 µl 5 µl – 1 µl C 4 µl 4 µl 1 µl 1 µl 3. Set the micropipettor to 4 µl, and add Solution I to each reaction tube. 4. Use a fresh tip to add the appropriate volume of Solution II at a clean spot on reactions tubes A, B, and C. Eject used tips into a small waste tip beaker. 5. Use fresh tip to add the indicated volumes of Solutions III and IV to the appropriate tubes. 6. Close the tops. Pool and mix the reagents by placing the tubes in a balanced configuration in a microcentrifuge and running for a few seconds. 2 TECHNIQUES IN MOLECULAR BIOLOGY – BASIC TECHNIQUES: PIPETING AND STERILE CULTURE 7. Each tube should have 10 µl of reagents at the bottom. To check your pipetting technique, set the pipettor to 10 µl and carefully draw the solution into a new pipet tip. a. Is the tip barely filled? (large undermeasurement) or b. Does a small volume of fluid remain in the tube? (overmeasurement) c. Is there a small air space left in the tip? (undermeasurement). Measure the actual volume by rotating the volume adjustment knob downward until the fluid reaches the tip, and read the indicated volume. 9. If one or more measurements is inaccurate, repeat the exercise and check results again. II. Practice with Medium- and Large-volume Micropipettors This exercise simulates the pipetting done in, for example, a bacterial transformation or plasmid DNA preparation for which 50 – 1000 µl pipettor is used. It is much easier to mismeasure with a large volume pipettor – especially the P1000 type. It is also easy to draw fluid into the barrel of the pipettor accidentally. The plunger must be released slowly when pipetting. 1. Label two 1.5 ml microtubes E and F. 2. Use the matrix below as a checklist while adding solutions to each reaction tube. Use the techniques discussed above for pipetting. Add the volumes up to see if the tubes will be balanced for centrifuging when you Tube Soln. I Soln. II Soln. III Soln. IV have completed pipetting. Use the pipettor for which the range best matches E 45 µl 155 µl 250 µl 550 µl the volume to be pipetted. F 30 µl 170 µl 375 µl 425 µl 3. Pipet the volumes as before, using a fresh tip when appropriate (i.e., you may use the same tip for the first pipetting of the same reagent into clean tubes. After that, you must use a new, clean tip for each pipetting). 4. Briefly spin down all the reagents in your microcentrifuge. 5. Set your P1000 to the appropriate volume to measure the total you determined for each reaction tube. Slowly draw the fluid into the pipet tip. Ask the same questions (a, b, c) in exercise I. 7. above, and repeat measurements if any of your pipetting was inaccurate. 3 TECHNIQUES IN MOLECULAR BIOLOGY – BASIC TECHNIQUES: PIPETING AND STERILE CULTURE III. Practice Sterile Use of a 10 ml Serological Pipet The key to successful sterile technique at the bench is to work quickly and efficiently. Before beginning, clear off the lab bench and arrange tubes, pipets and culture media within easy reach. You may want to simulate the sequence of events of taking off and replacing caps without setting them down before you proceed. If you believe at any time that you may have compromised sterility, assume that you have and replace any plastic or media with reliably clean items. As a general rule of thumb, use sterile technique if live bacteria are needed at the end of the manipulation (e.g., general culturing, transformation). Sterility is not required if bacteria are being destroyed in the experiment (e.g., after an overnight culture, isolating bacteria for DNA isolation). Cleanliness, of course, is always desirable. When in doubt, use sterile technique. 1. Loosen caps on any tube or media container (sterile culture tube, container of sterile LB broth) so they can be easily lifted off. 2. Open the end of the pipet sleeve and attach the pipet bulb. Squeeze the bulb so that it is prepared to suck. Remove the sleeve from the pipet. 3. Lift the lid from the LB broth container (do not set down), insert the pipet and withdraw 5 ml of broth into the pipet. 4. Replace the LB broth container lid loosely. 5. Remove the lid from the culture tube (do not set down), and transfer the LB broth to the tube. Replace the lid, with the lid on the ‘fingers.’ 6. Place the tube in a rack in the 37° C incubator to test its sterility (you may do this also with your LB broth container). The broth should be clear tomorrow if sterility was maintained. Check and record in your lab book. Place your tube (after one day of 37oC) into the refrigerator for later use. 4 TECHNIQUES IN MOLECULAR BIOLOGY – BASIC TECHNIQUES: PIPETING AND STERILE CULTURE Basic Techniques – Bacterial Culture I. Isolation of Individual Colonies by Streaking Good bacteriological technique dictates that any culture is begun with a single well-isolated bacterial colony, which represents a clone – the progeny of a single bacterium. This way one is reasonably certain that the culture grown contains a single, isogenic entity (all the same genotype), and not more than one kind of bacteria or other microbe. This exercise teaches you how to make a bacterial culture plate that has such isolated bacterial colonies. This exercise also demonstrates the phenomenon of antibiotic resistance by comparing the growth of two different strains of bacteria – one with an ampicillin resistance gene on a plasmid, and one without – on culture plates with or without the antibiotic ampicillin.

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