Microbial Genetics—Use of micro-pipettors to transfer accurate volumes of solutions.
Introduction: During the previous lab you learned how to correctly dial pipettors to pick up
and expel a given volume of solution. You also learned how to use the uptake and expel
button at the top of the pipettor to accurately transfer solutions.
During this lab session you will learn how to accurately pipette dilute solutions and
viscous solutions. Furthermore you will learn how to pipette solutions into small receptacles
such as micro-centrifuge tubes and agarose gel wells.
Laboratory exercises
A. The first exercise involves using the P1000 pipette to pipette 1 mL or 1000 uL of water
into a tray. This tray will be weighed to determine if you have properly transferred 1 mL of
water to the weighing tray (1 mL of distilled water at room temperature weighs 1 gram).
1. Obtain a tray and place it on the weighing pan of the scale. Tare the scale such
that the readout reads zero.
2. Pipette 1 mL of the provided water into the tray. The instructor will determine if
you have transferred the correct volume.
a. If you pipette the correct volume, the instructor will ask you to exchange
your old tip for a new tip and to repeat the transfer process. If you do this
correctly you may sit down.
b. If you do not pipette the correct volume you should go to the end of the line
and practice pushing the pipette button. You will get as many trial times as
required until you have mastered this exercise.
B. The second exercise involves using the P1000, P200 and P20 pipettors to transfer a given
amount of solution from one container to another. The instructor will place a predetermined
volume of solution in a container and ask you to transfer a given volume to another container
for a given number of times. The instructor and the student will determine if the correct
amount has been transferred by determining how much solution is left in the original
container.
1. P1000 (Purple pipettor): transfer 1 mL of the solution from the original container
to the new container 4 times. Use a new pipette tip each time. (Why?) Save the
original container and show this to your instructor.
2. P200 (Pink pipettor): transfer 200 uL of the solution from the original container to
the new container 5 times. Use a new pipette tip each time. Save the original
container and show this to your instructor.
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3. P20 (Red pipettor): transfer 10 uL of the solution from the original container to
the new container 5 times. Use a new pipette tip each time. Save the original
container and show this to your instructor.
C. The third exercise involves learning to pipette dilute solutions and viscous solutions and
learning to pipette these solutions into a small receptacle. To this end each student will mix a
small volume of DNA loading buffer (viscous) into a small volume of dilute buffer. Finally
the students will transfer the mixture to a small well found in an agarose gel.
Dilute solutions contain more water than solutes and therefore are easier to pipette
accurately. Viscous solutions such as DNA loading buffer contain large concentrations of
sugars and salts (Why?) and are somewhat more difficult to pipette accurately.
1. Obtain three small microfuge tubes from the instructor. One tube (tube 1) contains
a predetermined amount of dilute buffer solution. One tube (tube 2) contains a
predetermined amount of the viscous 6X DNA loading buffer. The final tube (tube 3)
is empty.
a. Pipette 10 uL of the dilute buffer to the empty tube (tube 3)
b. Pipette 2 ul of the 6X DNA loading buffer into tube 3 and mix with the
dilute solution. The instructor will show you how to do this.
c. Finally pipette 12 ul of the mixed solution to a well in an agarose gel. The
instructor will show you how to do this.
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