Measurements and Micropipetting

 

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Measurements and Micropipetting

 

     This laboratory introduces micropipetting techniques that are used in many of the experiments you will perform during this workshop.  Mastery of these techniques is important for good results in the experiments that will follow.  Most molecular biology and microbiology laboratories are based on microchemical protocols that use very small volumes of DNA and reagents.  These require the use of an adjustable micropipettor that measures as little as one microliter (ml), one millionth of a liter!

     Become familiar with metric units of measurement and their conversions.  We will concentrate on liquid measurements based on the liter, but the same prefixes (milli and micro) also apply to dry measurements based on the gram.  The two most useful units of liquid measurement in molecular biology are the milliliter (mL) and the microliter (ml).

1 mL = 0.001 liter                    1,000 mL = 1 liter

1 ml = 0.000001 liter                 1,000,000 ml = 1 liter

    

     A digital 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 farther 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  The withdrawn fluid is then expelled by depressing the plunger again.

     The volume range of a digital micropipettor varies from one manufacturer to another.  A small volume micropipettor (with a range of 0.5–10 ml or 1-20 ml) and a large volume micropipettor (100-1,000 ml) are used most frequently.  A mid range micropipettor (10-100 ml or 20–100 ml) is used less frequently.

Familiarize yourself with small scale and large scale micropipettors that you will be using in this laboratory.  A P20 micropipettor measures volumes between 2-20 ml.  Locate the digital readouts on your micropipettors.  It is located below the plunger.  The digital readout has three number boxes that display the specified volume.

   2

   0

   0


 The above digital readout indicates a reading of 20 ml on a 2-20 ml volume micropipettor.

   0         

   1

   0

The above digital readout indicates a reading of 10 ml on a 2-20 ml volume micropipettor.

 1

 2

 5

The above digital readout indicates a reading of 12.5 ml on a 2-20 ml volume micropipettor.

 2

 0

 0

The above digital readout indicates a reading of 200 ml on a 20-200 volume micropipettor.

 0

 2

 3

The above digital readout indicates a reading of 23 ml on a 20-200 volume micropipettor. .

 1

 0

 0

The above digital readout indicates a reading of 1000 ml or 1 mL on a 100-1000 ml micropipettor.

 5

 2

 0

The above digital readout indicates a reading of 520 ml on a 100-1000 ml micropipettor.

 

     Take the following precautions when using a digital micropipettor:

·  Never rotate the volume adjustor beyond the upper or lower range of the pipet, as stated by the manufacturer.

·  Never use the micropipettor without the tip in place; this could ruin the piston.

·  Never invert or lay the micropipettor down with a filled tip; fluid could run back into the piston.

·  Never let the plunger snap back after withdrawing or expelling fluid; this could damage the piston.

·  Never immerse the barrel of the micropipettor in fluid.

·  Never flame the tip of the micropipettor.

·  Never reuse a tip that has been used to measure a different reagent.

 

I.                   General Use of Digital Micropipettors

 

1.      Rotate the volume adjustor to the desired setting.  Note the change in plunger length as the volume changes.  Be sure to properly locate the decimal point when reading the volume setting.

2.      Firmly seat a proper-sized tip on the end of the micropipettor.

3.      When withdrawing or expelling fluid, always hold the tube firmly between your thumb and forefinger.  Hold the tube nearly at eye level to observe the change in the fluid level in the pipet tip.  Do not pipet with the tube in the test tube rack.  Do not have another person hold the tube while you are pipetting.

4.      Each tube must be held in the hand during each manipulation.  Open the top of the tube by flipping up the tab with your thumb.  During manipulations, grasp the tube body (rather than the lid), to provide greater control and to avoid contamination of the mouth of the tube.

5.      For best control, grasp the micropipettor in your palm and wrap your fingers around the barrel; work the plunger (piston) with the thumb.  Hold the micropipettor almost vertical when filling it.

6.      Most digital micropipettors have a two-position plunger with friction “stops”.  Depressing to the first stop measures the desired volume.  Depressing to the second stop introduces an additional volume of air to blow out any solution remaining in the tip.  Notice these friction stops; they can be felt with the thumb.

7.      To withdraw the sample from a reagent tube:

a.       Depress the plunger to first stop and hold it in this position.  Dip the tip into the solution to be pipetted, and draw fluid into the tip by gradually releasing the plunger.  Be sure that the tip remains in the solution while you are releasing the plunger.

b.      Slide the pipet tip out along the inside of the reagent tube to dislodge any excess droplets adhering to the outside of the tip.

c.       Check that there is no air space at the very end of the tip.  To avoid future pipetting errors, learn to recognize the approximate levels to which particular volumes fill the pipet tip.

d.      If you notice air space at the end of the tip or air bubbles within the sample in the tip, carefully expel the sample back into its supply tube.  Coalesce the sample by sharply tapping the tube on the bench top or pulsing it in a microfuge.

8.      To expel the sample into a reaction tube:

a.       Touch the tip of the pipet to the inside wall of the reaction tube into which the sample will be emptied.  This creates a capillary effect that helps draw fluid out of the tip.

b.      Slowly depress the plunger to the first stop to expel the sample.  Depress to second stop to blow out the last bit of fluid.  Hold the plunger in the depressed position.

c.       Slide the pipet out of the reagent tube with the measurement plunger depressed, to avoid sucking any liquid back into the tip.

d.      Manually remove or eject the tip into a beaker kept on the lab bench for this purpose.  The tip is ejected by depressing the plunger beyond the second stop.

9.      To prevent cross-contamination of reagents:

a.       Always add appropriate amounts of a single reagent sequentially to all reaction tubes.

b.      Release each reagent drop onto a new location on the inside wall, near the bottom of the reaction tube.  In this way, the same tip can be used to pipet the reagent into each reaction tube.

c.       Use a fresh tip for each new reagent to be pipetted.

d.      If the tip becomes contaminated, switch to a new one.

 

Practice with Small-Volume Micropipettor

 

This exercise simulates setting up a reaction, using a micropipettor with a range of 0.5 -10 ml or 1–20 ml.

 

  1. Use a permanent marker to label three 1.5 mL microfuge tubes A, B and C.
  2. Use the matrix below as a checklist while adding solutions to each microfuge tube.

 

Tube                Sol. I               Sol. II              Sol.III             Sol. IV

A                     4 ml                   5 ml                   1 ml                   ____

B                      4 ml                   5 ml                   ____                1 ml

C                     4 ml                   4 ml                   1 ml                   1 ml

 

  1. Set the micropipettor to 4 ml, and add Solution I to each tube.
  2. Use a fresh tip to add the appropriate volume of Solution II at a clean spot on tubes A, B and C.
  3. Use a fresh tip to add 1 ml of Solution III to tubes A and C.
  4. Use a fresh tip to add 1 ml of Solution IV to tubes B and C.
  5. Close the tops of the tubes. 
  6. Pool and mix the reagents by placing the tubes in a microfuge and apply a short pulse of several seconds  Make sure that the tubes are placed in a balanced configuration in the microfuge rotor.  Spinning tubes in an unbalanced position will damage the microfuge motor.
  7. A total of 10 ml of reagents was added to each tube.  To check the accuracy of your measurements, set the pipet to 10 ml and very carefully withdraw the solution from each tube.
    1. Is the tip barely filled?

 

OR

 

    1. Does a small volume of fluid remain in the tube?  This indicates an overmeasurement.

 

OR

 

    1. After withdrawing all fluid, is an air space left in the end of the tip?  This indicates an undermeasurement.  (The actual volume of fluid can be determined by simply rotating the volume adjustment to expel air and push fluid to the very end of the tip.  Then, read the volume directly.
  1. If several measurements were inaccurate, repeat the exercise to obtain nearly perfect results.

 

III.  Practice with Large-Volume Micropipettor

 

This exercise simulates a bacterial transformation or plasmid preparation, for which a 100-1000 ml micropipettor is used.  It is far easier to mismeasure when using a large-volume micropipettor.  If the plunger is not released slowly, an air bubble may form or solution may be drawn into the piston.

 

  1. Use a permanent marker to label two 1.5 microfuge tubes E and F.
  2. Use the matix below as a checklist while adding solutions to each tube.

 

Tube                Sol. I               Sol. II              Sol. III            Sol. IV

E                      100 ml               200 ml               150 ml               550 ml

F                      150 ml               250 ml               350 ml               250 ml

 

 

  1. Set the micropipettor to add appropriate volumes of solutions I-IV to tubes E and F.  Follow the same procedure as for a small-volume pipettor.
  2. A total of 1,000 ml of reactants was added to each tube.  To check the accuracy of your measurements, set the micropipettor to 1,000 ml and carefully withdraw the solution from each tube.

 

    1. Is the tip barely filled?

 

OR

 

    1. Does a small volume of fluid remain in the tube?

 

OR

 

    1. After withdrawing all fluid, is an air space left in the end of the tip?

 

  1. If your measurements were inaccurate, repeat the exercise to obtain nearly perfect results.


RESULTS AND DISCUSSION

Inaccurate pipeting is a chief contributor to poor laboratory results.  If you are still umcomfortable with micropipettors, take the time now for additional practice.  This technique will soon become second nature to you.

  1. Why must tubes be balanced in a microfuce rotor?
  2. What common error in handling a micropipettor can account for pipetting too much reagent into a tube?  What errors account for underpipetting?