Animal Laboratory - Pipetting Accuracy Lab Form (1)
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Animal Physology: Pipetting Accuracy Lab
Group Members:
Experimental Design
What questions are being asked in this exercise? What experimental design do you think would allow you to answer your question(s)? Describe what you expect you will find? In other words describe what you expect your result will be. This exercise requires you to connect concepts from your general chemistry (concentration/dilutions) and general mathematics (equations for curves/particularly a straight line).
1.
Lab Safety accoutrements (lab coats, gloves, eyewear, shoes that cover foot, etc.)
2.
Serological 3.
Pipettes (10ul, 20ul, 1000ul)
4.
Pipette tips that fit 10ul, 20ul, 1000ul.
5.
Eppendorf tubes
6.
Test tube rack
7.
Spectrophotometer that can read at 595nm
8.
Bradford reagent
9.
Bovine albumin
10.
Weigh boats
11.
Weigh scale
12. Spatula
13.
Stir bars
14.
Stir plate
What you will need
Mission:
This lab is designed to give you practice in pipetting small volumes. You will be required to pipette various designated volumes of water into Eppendorf microcentrifuge tubes. You will employ two approaches to assess your accuracy in pipetting:
1.
Correlate pipetted volume to actual volume.
2.
Reproducibility of pipetted volume using Bradford assay.
9/25/2023
Question #1:
Am I pipetting the amount I am seeking to pipette? Or put another way Am I actually pipetting 1ml or .5ml or 0.2ml, etc.?
Experimental design: compare your pipetted volume to a known or reference volume that you trust to be accurate. In this case we will weigh out 0.2ml, 0.5ml, 1ml, 2ml, and 5ml.
Volume (ml)
Method
0.2
0.5
1
2
5
Measured weight volume Micropipette volume
Note: this method assumes 1ml of water = 1gm. This is a reasonable assumption because water is considered as having very little density to consider.
Question #2:
Is my pipetting reproducible? In other words, can I consistently pipette the same volume every time when the pipette is set at .2ml or 1ml, or .1ml, etc.?
Experimental design: Make 10 mls of a stock solution of albumin with a concentration of 2mg/ml. From this stock solution prepare a .1mg/ml solution, 0.2mg/ml, 0.5mg/ml. This should result in you having 4 tubes containing the following concentrations of albumin: .1, .2, .5, and 1mg/ml.
Use a colorimetric assay that measure protein concentration to determine if you do have these concentrations in your tubes.
Perform the assay twice to see if your results are very close. If they are then your pipetting is consistent or reproducible.
Question #3
Is there a difference in volume pipetted between serological pipettes and micropipettes? Sometimes you will have to come up with plan B if you find you do not have all of the supplies or equipment you need. So, it is good to have a second way of pipetting, particularly since micropipettes cost about $2,000 each. Serological pipettes cost about 20 cents each. These are not as accurate but you can upscale the assay and still perform the experiment. So here we can compare how closely the serological method Question #1:
You need beaker, water, weigh boats, Eppendorf tubes, tube rack, pipettes, pipette tips, and weigh scale.
Expected true volumes measured
1.
Go to weigh scale and weigh out 0.5, 1, 2, and 5 ml volumes. 2.
To do this place a weigh boat on the scale. 3.
Tare out the weight of the weigh boat. 4.
Using a eye dropper add water to the weigh boat until you get to the desired weight (0.5 g, 1g,2 g, and 5 g). 5.
Using a graduated syringe aspirate the water from the weigh boats up into the syringe to determine if weight of water really does equate to volume of water.
6.
Prepare a spreadsheet to enter your data.
NOTE: Science observations reveal that 1 cubic centimeter (cc) or 1 ml of water equals 1g.
Pipetted Volume
1.
Place 16 tubes in the rack (two rows with 8 tubes each).
2.
Label row one tubes as .5,1, 2, and 5 mls. You should have two tubes labeled as 0.5, two tubes as 1, two Experimental Protocol
Describe the step-by-step procedure you will follow to execute your design.
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Question #1 (cont’d): Bradford Assay. This assay uses a dye (Coomassie) to bind to protein. When the dye binds to a protein the color of the dye changes from a blue that absorbs at the wave length 470nm to a color that absorbs at 595nm. So, the more
protein is in the protein solution the more light is absorbed at 595nm. Clever individuals capitalized (I mean this because millions of dollars are earned buying this reagent) on two principles: 1) the effect of Coomassie binding to proteins, and 2) and the principle of a straight line from mathematics. The principle of a straight line allows for us to determine the concentration of an unknown. The equation of a straight line is y = mx + b. Therefore if we can make a straight line plot where x is the concentration and y is absorbance, then we can determine the concentration of our experimental sample if we know the absorbance. We do this simply by creating a straight line where we know the slope (m), y intercept (b), absorbance (y). Solving for x will tell us the concentration of our unknown sample.
1.
Any colorimetric assay that is used to determine concentration of unknown samples requires the development of a standard curve. The curve could be any shape that has equation for only one x value. The most common standard curve shape is a straight line where y=mx+b.
2.
To make a standard curve you prepare standards where you know the concentrations. You will need a scale, weigh boat, albumin, rack, tubes, beaker, water, spatula
a.
prepare 10 mls of a stock solution of albumin that has a concentration of 2mg/ml. b.
prepare 2 mls of 0.1 mg/ml concentration using the stock solution
c.
prepare 2 mls of 0.2 mg/ml concentration using stock solution
d.
prepare 2 mls of 0.5mg/ml concentration using the stock solution
3.
Label a set of tubes 0, .1, .2, 0.5, and 1 mg/ml. Label a second set with the same labels. Again we are doing them in duplicates.
4.
Pipette 40 ul of each standard into the appropriately labeled tubes.
5.
Then pipetted 2ml of the Bradford reagent to each tube.
6.
Let sit at room temperature for 5 min before reading absorbance.
7.
Transfer solution to cuvette tube. Place cuvette in spectrophotometer and read the absorbance. Be sure to
record your results.
8.
Discard cuvette in regular trash.
Once you have collected the data open MS Excel so you can perform a correlation analysis. A correlation analysis allows you to determine how strongly two things are related. For this analysis make expected volume the x axis and the actual volume the y axis. The idea is to determine if your pipetted volume (actual) is the same as what the expected volume was. The stronger the correlation the more closely your pipetting is to the expected volume.
Tube #
Desired Vol
(mls)
Pre Water Ependorf Wt Post water Eppendorf Wt
Expected Vol
Pre-water eppendorf
Micropipette
Post-water eppendorf
Micropipette
Actual Vol
Results
Question #1
: Provide the raw data results from your procedure/protocol. This is usually in tabular form. You can use a spreadsheet such as MS Excel and copy it into this section. Next you will need to plot your results. The type of plot is often dictated by the question you are asking. What type of plot is best for your research question? Be sure to insert them in this document.
Question #2: Bradford Assay standard curve.
Enter the absorbance values for each of your standard including the duplicates. Once you have all of the absorbance values for all your standards (plus duplicates) use the MS Excel spreadsheet to plot the known standard concentrations on the x axis and the absorbance values on the y axis. Have Excel calculate equation for a straight line of the standard curve and the r
2
value for the scatter plot. Use your phone to determine the square root of r2 to get the r value. The r value tells you how strong the x is related to y. In other words, it shows how likely changes in one variable will be accompanied by a change in the other variable. The dependent variable y is strongly dependent upon the change in independent variable x when you have high r values. For my research I require my standard curves to have an r value of 0.95 or higher. On the other hand, r
2
tells us how much of the change in the dependent variable (y) is due to a change in the independent variable (x).
Item #
Standard [ ]
mg/ml
Absorbance tube
A
Absorbance Tube
B
Blank
0
0.1
0.2
0.5
1
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Interpretation
Do your results meet your expectation? Yes or no and explain. Do your results provide an answer to your research questions? What was (were) the answers that your results support?
INSERT RESPONSE HERE
Conclusion
Now that all is said and done what do you make of everything you have just done in this lab?
INSERT RESPONSE HERE
INSERT RESPONSE HERE
INSERT RESPONSE HERE
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