Lab 2: PROCEDURE Part 2

PIPETTE PRACTICE

After discussing safety protocols, you will do a short exercise to familiarize yourself with the pipettes you will be using this semester. Once you have completed the pipette exercise, you will set up your PCR reactions.

Micropipettes

You have four micropipettes to work with:

• the p1000 is used for volumes of 100–1000 μL,
• the p200 is used for volumes 20-200 μL or p100 is used for volumes of 10–100 μL,
• the p20 is used for volumes of 2–20 μL,
• and the p10 is used for volumes of 0.5–10 μL.

The pipettes are most accurate at the higher end of their range, so even though the p100 could be used to pipette 10 μL, the p20 would be more accurate.

Pipette Tips

The blue tips are for the p1000, the yellow tips are for the p100, p200 and p20, and the clear tips are for the p10.

When you empty a tip box, you must refill it with new tips; you may then exchange it for a full, sterile box from your TA. Points will be deducted from your lab performance score if you do not do so.

When not in use, keep your tip boxes closed for sterility.

When you are done with tips, please discard them in the large cardboard boxes labeled TIP WASTE. Nothing else should be put in this box—please discard used gloves and paper towels in the regular trash. If you have a question about where something should be discarded, please ask your TA.

Exercises: Micropipettes

• Find an Eppendorf tube of Coomassie Brilliant Blue dye. (Be careful—this solution stains clothing.)

• Practice pipetting large volumes by creating serial dilutions (it is ok to do this with a partner)

• • Make 500 μL final volume of the following serial dilutions of the blue dye (tap water is fine for diluting and we recommend using 1.5 mL microfuge tubes):
    • 1/2
    • 1/4
    • 1/8
    • 1/16
    • 1/32
    • 1/64
    • 1/128
  • Pipette 100 μL of each solution into the wells of a 96-well plate in triplicate.

• Practice pipetting small volumes (it is important for everyone to do this individually)

• • Pipette 99 μL water into each of three wells in your plate
  • Add 1 μL of the blue dye (from the original stock). Pipetting such a small volume can be tricky; here are a few suggestions:
    • Be sure your pipette tip gets down into the blue dye. After drawing up the 1 μL, look at the pipette tip. You should be able to see a small amount of liquid in the end of the tip.
    • Be sure the tip gets down into the solution you are adding the 1 μL to (in this case the water in your 96-well plate). Eject to the second stop, and then draw your pipette tip out. Look at the tip and be sure it is now empty.
  • Mix by pipetting up and down with the larger pipette
• Measure the absorbance of the plate at 595 nm (A₅₉₅). Be sure to add a blank. Once you have the data, take a look at the triplicates and see how precisely you pipetted the solution into the plate. Next, use the absorbance measurements to determine how accurate your dilutions were.