Using Micropipettors and Creating Dilutions PDF

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CHEM 51LD Volumetrics and Using Micropipettors USING A MICROPIPETTE: Micropipettors vary by manufacturer, but most are based on a popular model called the Pipetman™. They are used for dispensing microliter volumes accurately.

P10 0.5-10 microliter P50 5-50 microliter P20 20-200 microliter P100 100-1000 microliter Directions: 1. Set the desired volume by turning the centrally located rings clockwise to increase volume or counterclockwise to decrease volume. 2. Push the micropipettor into a tip that is held in a tip rack until the tip is firmly seated on the pipettor. Never touch a disposable pipette tip with your fingers. 3. To pick up liquid, displace the appropriate amount of air from the tip by pushing the plunger to the first resistance point and hold the plunger there. 4. Immerse the tip in the solution to be transferred. Do not push the tip against the bottom of the container. 5. SLOWLY release the plunger, drawing liquid into the tip. If you release the plunger too quickly, the liquid will spray up into the pipettor and damage it. Be extra slow with viscous liquids, which are drawn very slowly into the tip. 6. To dispense the liquid, touch the pipette tip to the wall of the tube and slowly depress the plunger to the first stop. After about a second, when the liquid stops flowing, depress the plunger all the way to dispense the remaining bit of liquid. Make sure the last drop sticks to the side of the tube and not the pipette tip.

7. Remove the tip from the vessel and hold it over the appropriate waste container. Press hard on the tip-ejector button to expel the used tip.

Tips for Using Micropipettors: ■ Never exceed the upper or lower limits specified of these expensive pipettors. ■ Whether you need sterile conditions or not, never allow the pipette tip to touch anything other than the solution you are transferring. ■ When transferring liquids, hold the micropipettor in a vertical or near-vertical position.

MAKING DILUTIONS: Dilutions are made by taking a concentrated aliquot from a known concentration of stock solution and diluting it with a solvent. M1V1 = M2V2 M1 = concentration of stock solution V1 = volume of aliquot of stock solution M2 = concentration of new diluted solution V2 = total volume of new diluted solution

Independent Dilutions from Stock Solution This technique is usually used when the stock solution is in abundance. To make diluted solutions directly from the stock solution a specific aliquot is taken from and then diluted with solvent to the desired concentration to give the final volume.

10 µ L

1.0 X 10 5

20 µ L

1.0 X 10 4 2.0 X 10 4

30 µ L

3.0 X 10 4

40 µL

4.0 X 10 4

50 µL

5.0 X 10 4 Total volume: 10 mL

Serial Dilutions from Stock Solution This technique is usually used when the stock solution is not in abundance or if certain volumes are difficult to measure. To make serial diluted solutions a large amount of aliquot is taken from the stock solution and a initial dilution is made by adding solvent to give the desired concentration. In order to obtain less concentrated solutions, subsequent aliquots are taken from each previous solution. This concentrated aliquot is then diluted using solvent to give the final volume.

100 µ L 50 µL

1.0 X 10 5

1.0 X 10 4

40 µL

5.0 X 10 3

20 µL

2.0 X 10 3

10 µ L

4.0 X 10 2

4.0 X 10 1 Total volume: 10 mL

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MICROPIPETTOR)PRACTICE)ASSIGNMENT) ) Before!you!will!be!allowed!to!use!a!micropipettor!for!an!experiment,!you!will!need! to!demonstrate!to!your!TA!that!you!can!successfully!pipette!water.!!Practice! pipetting!water!using!any!of!the!micropipettors!available.!!When!you!are!ready,! demonstrate!the!following!to!your!TA.! 1. Set!a!volume!in!the!middle!of!the!range!for!the!micropipettor!you!are!using.!! Show!your!TA!the!volume!you!have!set!on!the!micropipettor.! 2. Place!a!disposable!tip!on!the!micropipettor.! 3. Transfer!the!appropriate!volume!of!water!from!one!container!to!another.! 4. Adjust!the!volume!setting!on!your!micropipettor!to!a!volume!on!the!high!end! of!its!range.!!Show!your!TA!the!volume!you!have!set.! 5. Transfer!the!appropriate!volume!of!water!from!one!container!to!another.! 6. Adjust!the!volume!setting!on!your!micropipettor!to!a!volume!on!the!low!end! of!its!range.!!Show!your!TA!the!volume!you!have!set.! 7. Transfer!the!appropriate!volume!of!water!from!one!container!to!another.! 8. Eject!the!disposable!tip!from!the!micropipettor.! ! When!you!have!successfully!demonstrated!all!of!the!tasks!listed!above!to!your!TA’s! satisfaction,!you!are!approved!to!use!micropipettors!in!future!experiments.!!Be!sure! to!refer!to!this!handout!and/or!ask!your!TA!if!you!are!ever!unsure!on!how!to!use!a! micropipettor!!...