Reference no: EM132821756

BC2013 Principles of Biochemistry - James Cook University

Practical - Pipetting Skills, Precision and Accuracy

Learning outcome 1: To practice using micropipettes
Learning outcome 2: To perform dilutions and represent measured results graphically
Learning outcome 3: To explain error in terms of precision and accuracy

Question 1. Since we are consistently halving the concentration of dye with each dilution, what do you expect the ideal shape of absorbance vs. concentration to be?

Question 2. Which experiment do you think will give more accurate results? Which one do you think will give more precise results? Explain.

Procedure

Part 1: Parts of the pipette

Each student will have 1 ml, 200 μl and 20 μl pipettes

1. Look at the tops and sides of each of the pipettes

a) What is the range of each pipette?
b) Which tips should be used with each pipette?
c) Practice pressing the button, feel for the first and second stops. What is the extra air at the second stop for?

d) What can happen if you over-wind pipettes (in either direction)?

2. Using some water in a microfuge tube and a weigh boat, practice aliquotting water into the weigh boat.

e) To aspirate a liquid, press down to the FIRST STOP ONLY, and immerse the tip just below the surface of the liquid.
f) When expelling the liquid, press down to the second stop, the extra air will ensure that all the liquid is expelled.
g) Touch the end of the tip lightly on the side of the vessel so that the surface tension of the water will ensure smooth transfer from the tip.
h) Practice sucking the liquid in and out gently to stir the water, this is how solutions are mixed.

Part 2: Weighing volumes of distilled water

In this exercise you can get an idea of the sorts of error that you can expect when using pipettes. The maximum allowable deviations that are accepted in calibrated pipettes at room temperature and atmospheric pressure are:

100-1000 ul ± 0.8%
10 ul ± 1.2%
5 ul ± 2.5%

Notice that the error is greater when a pipette is set to its minimum volume, so it is always better to use a pipette as close to its maximum range as possible.

1. Place a plastic weigh boat on the pan of a 3- or 4-place balance, zero (tare) the balance.
2. Take the 1 ml pipette and some distilled water. Set the pipette to 1000 μl and carefully transfer one aliquot of water to the weigh boat. Record the weight in the table provided in the answer sheet.
3. Repeat this 2 times, record the weight each time (Table 1).
4. Repeat these steps for 200 μl and 20 μl pipettes, each set to maximum volume
5. Calculate the following and record in the answer sheet provided:
a) Mean weight of the 3 aliquots and this value as a percentage of what the weight should be (don't forget units, if you are unsure ask a tutor)

b) The maximum measured value as a percentage of the nominal weight.

c) The minimum measured value as a percentage of the nominal weight.

These values give you an idea of the errors that YOU are introducing when you are pipetting. The pipettes in the lab have been calibrated and if used correctly should be within the limits of error. Please remember, as with most other practical components you do in the lab, accurate pipetting is a skill, the more you do it the more accurate you become. The final practical will be a practical exam which will test how accurate your pipetting is, because of this we recommend that you DO NOT just let you partner do all the pipetting during the semester.

Part 3: Precision and accuracy in dilutions

A stock solution of blue dye dissolved in water and a microtitre plate will be supplied. Divide the microtitre plate up as follows:

Experiment 1

1. Pipette 200ul of your stock into each of wells A1, A2, and A3.
2. Pipette 100ul H2O in the remaining wells B1 through to H3
3. Perform serial doubling-down dilutions by pipetting 100ul from A1 into B1, mix several times by gentle pipetting and then remove 100ul into C1, repeat until you reach H1. Mix this last row thoroughly and then remove 100ul into the waste bucket. Repeat the procedure with columns A2 - H2 and then A3 - H3.

Experiment 2

Now we will repeat the process in columns 4, 5, and 6 but instead of doing doubling-down dilutions, will do each dilution individually. You will find that after your 1/32 dilution the volumes become too small to measure with a p20, therefore experiment 2 only uses rows A to F.

1. Refer to Table 2 in your answer sheet. Given that the final volume of each well must be 100
μl and that the amount of dye stock added will be halved each time, calculate the volumes of dye and water to be added to each row.
2. Add the volumes of water specified in Table 2.
3. Now to tube A4-6 add 100 μl of dye solution to each of wells A4 to A6.
4. Continue to add an appropriate amount of dye stock to tubes B4 through F6 so that the final volume in each well is 100 μl.
5. When your plate is completed a tutor will show you how to read the plate using a plate reader and an absorbance of 450 nm.
6. While you are waiting for your results you can do Part 1 of the prac, or go on to the next section and draw your graph.
7. Place 100 μl of water in wells H4-6, these will be used as the blank for the experiment.

Attachment:- Practical - Pipetting.rar