01-Lab Fundamentals - IN CLASS DOCUMENTS

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Biology

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Dec 6, 2023

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5STATION #1: How to Use a Micropipette Scientists routinely measure various volumes of liquids. The equipment used depends upon the volume being measured and the accuracy required. For example a 100 – 1000  l adjustable volume pipet can measure 1 ml of liquid accurately, while a transfer pipet can measure the same volume but with much less accuracy. This laboratory exercise involves measuring and transferring various volumes of liquid with the proper equipment and then determining the accuracy of the liquid transferred. Goals of lab exercise  Learn the use of pipets. o accurately measure volumes using a serological pipet o accurately measure volumes using adjustable-volume micropipets o accurately measure volumes using a 1 ml graduated transfer pipet  Calculate percent error of single measurements  Calculate mean and standard deviation among measurements How to use a Micropipette Parts: 1 . Volume adjustment dial 2 . Tip ejector button 3 . Plunger button 4 . Stainless steel micrometer 5 . Digital volume indicator 6 . Stainless steel ejector arm (removable) 7 . Plastic shaft 8 . Disposable yellow or blue tip Pointers:  Never rotate the volume adjustment knob past range o The ranges are: P-10 – up to 10  l P-20 - up to 20  l - up to .02 ml P-200 - 20 to 200  l - up to .2 ml P-1000 - 200 to 1000  l - up to 1.0 ml  Never use a micropipet without a tip in place o this can ruin the inner workings  Never lay the micropipet down on its side when it contains liquid o this liquid could run into the micropipette  Never let the plunger snap back after withdrawing or ejecting fluid o this could damage the piston  Never immerse the barrel of the micropipet into fluid  Always use a disposable tip! NOTE: All Micropipettes are different. Check the top of the tip ejector button to find the volume range for a particular pipet.

STATION #1: How to Use a Micropipette Procedure 1. Discuss the video you watched before class with your group members. Briefly describe proper pipetting techniques. 2. There are five micropipettes labeled A through D at your station. Fill in the blanks with the correct volume each micropipette is set to dispense. BE SURE TO INCLUDE UNITS. (NOTE: You may reference the diagram to help you.) A B C D Sample pipet settings : 3. Practice using the micropipettes. --- Use the unlabeled micropipettes.  Place a small volume of water into a beaker.  Choose one of the micropipettes at the station  Check the top of the micropipette plunger button to see the ranges of volume associated with that specific micropipette.  To change volume: o Rotate the volume adjustment knob until the digital indicator reaches your desired volume. o Remember: do NOT turn the settings on the pipet past the recommended maximum value this will result in breakage of the pipet.  Firmly, place a disposable tip on the shaft of the pipet.  Press down the plunger to the First Stop. o NOTE: You will be able to push past this point, but there is enough resistance to stop the movement if you try to be aware of it. o This part of the stroke is the calibrated volume that you see on the digital micrometer.  Hold the pipet vertically and immerse the disposable tip into the water. o NOTE: It is only necessary to place the tip in to a depth of several millimeters.  SLOWLY release the plunger button, allowing the plunger button to return to its original position. o Do not allow the button to snap up!!!  Keep the tip in the solution while releasing the plunger to ensure that the full volume of the sample is drawn into the tip.  Withdraw the tip from the sample.  To dispense the sample: o Place the tip against the side-wall of the receiving tube o Push the plunger down to the first stop o Then depress the plunger to the second stop in order to expel any residual sample in the tip.  While the plunger is still pushed down, remove the pipet from the tube and allow the plunger to slowly return to its original position. o This ensures none of the sample is ‘sucked’ up back into the tip.  Discard the disposable tip by pushing the ejector button. (Be careful where you point.) 4. Practice testing the accuracy of your pipetting skills.  Using a digital balance you can determine how accurate your pipet is and how accurate you are at pipetting. The rule of thumb is that 1 g =1 ml. If you pipet 540 l μ of water onto a digital scale it should measure 0.54 g because 1 ml = 1,000 l; therefore 540 μ l = 0.54 ml = 0.54 g. μ  Practice pipetting water at different volumes into the weight boats provided. Fill in the table below (the first row is an example). Be sure to tare your weight boats BEFORE you measure your sample. Micropipette Set volume ( l) μ Weight (g) % Accuracy = [ ( weight x 1,000) ÷ volume] x 100 P20 15.0 0.012 [(0.012 x 1,000) ÷ 15] x 100 = 80% P200 32.0 P1000 547.0 5. How many of the samples above were less than 90% accurate? WHY? 6. Make sure to discard your liquid waste and place the weigh boat back on the balance. MOVE TO STATION #2 when instructor gives the signal. If you have extra time, keep practicing.

STATION #5: Loading Gels Agarose gels matrices are used in the separation of biomolecules (DNA, RNA and protein) by electrophoresis (electrical current through porous material). This station will allow you to practice loading of agarose gels. 1. Watch the video on how to load an agarose gel: http://www.youtube.com/watch?v=tTj8p05jAFM Take notes. 2. Make sure each of the following materials is present at your station: P20 P10 yellow tips for P20 thin and long tips for gel loading microcentrifuge tube rack 1% agarose gel in chamber w/buffer 1X gel loading dye light box with gel images gloves Biohazard trash receptacle 3. Using the P20 and yellow loading tips each member of your group should practice loading 10 l μ of 1X loading buffer into the agarose gel. Load up to 4 wells per group.  When you are finished make sure you dispose your tips & gloves into the biohazard bag. 4. The amount of agarose in the gel affects the distance DNA travels through the gel. Observe the figure below and answer the question. HOW does agarose concentration affects DNA migration? 5. When performing gel electrophoresis, it is common to run a DNA ladder in one of the wells to assess the size of your DNA samples in the gel. Here are some examples: A: B: C: Figure 1. Agarose matrix.

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