Biolab 1 - bio lab PDF

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bio lab...

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Measurement, Pipette Use, and the Standard Curve Objectives 1. Dilute a concentrated stock solution to a working concentration. 2. Accurately and precisely use pipettes in a laboratory setting 3. Use a spectrophotometer 4. Generate and evaluate a standard curve. 5. Use a standard curve to determine the concentration of protein in milk.

Safety 1. Closed-toe shoes must be worn 2. Long hair tied back 3. Goggles must be worn at all times while experiments are being conducted in the classroom. Even if you’re done. 4. Gloves should be worn while performing the experiments. 5. All pipet tips should be ejected into the tip waste bin on your bench. 6. All Bradford reagent should be poured into the acid waste container on the side bench. Remember to put the lid back on when you are done. 7. Rinse your plastic cuvettes in the sink and place them in the trash can in the front of the room.

Lab Activity 1: Proper pipetting technique Your Lab Instructor will demonstrate the proper technique for using a micropipettor. 1. When instructed following the demonstration, pick up a micropipettor (it does not matter what size) and place the correct size tip on it. 2. Depress the plunger with your thumb and become accustomed to the "first stop" and the "second stop". 3. Practice depressing the plunger to the first stop and s l o w l y releasing it back to the end stop. 4. When your Lab instructor comes to visit your table and instructs you to do so, pipet the volume of water requested by your instructor from the container on your table into a microcentrifuge tube. 5. Under no circumstances should you ever dial a micropipettor above or below its stated volume range! (Look at the knob—it will tell you the range!) Doing so will damage the pipettor and likely make it extremely unreliable. 6. Your instructor may request that you repeat this process until you get it right. It's ok! It's practice and you want to become proficient.

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Lab Activity 2: Using Standard Curves to Calculate Unknown Concentrations Both Part A and Part B should be set up and performed simultaneously. Ideally, perform the serial dilution and initial testing of the milk first, then set up the standard curve and measure the appropriate dilution of milk. You should measure your milk sample on the spectrophotometer immediately after you have measured your standard curve.

Part A-- the standard curve Complete Table 1, calculating the volume of BSA, Bradford Reagent and 0.15M NaCl necessary to make a 1.0 mL solution with the final concentration shown in each row. You will be required to hand in the completed table.

You should have Table 1 already filled in BEFORE LAB. 1. Turn on the spec to warm up. 2. Label your tubes (cuvettes) at the top of the tube with a sharpie. 3. To each cuvette, add Bradford Reagent first, then NaCl, and finally BSA. Put the tip of the pipette tip into the reaction when delivering small volumes to your cuvette. Remember to remove the tip from the reaction before releasing the plunger after delivery. 5. Mix well by carefully and gently flicking the cuvette. 6. Incubate the tubes for approximately 3--5 minutes at room temperature 7. While incubating, set the wavelength, λ (lambda), of the spectrophotometer to 595 nm. This may have already be done, but you should check that the instrument is set correctly. 8. Gently wipe the exterior of each cuvette with a Kimwipe. 9. Blank (zer o) the spec with….What blank should be used for measuring your BSA solutions in the spectrophotometer? 10. Measure the absorbance of each sample. Place the cuvette in the spectrophotometer in the slot for cuvettes and gently close the lid. DO NOT FORCE THE LID CLOSED! Wait for the numbers to stabilize, and record the value, then move on to the next tube. 11. Fill in Table 2 with the absorption measurements of your known BSA standard solutions.

Part B-- Dilutions and Determining the Concentration of the Unknown 1. Obtain a sample of milk from the TA and record the identity of the milk.

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2. Make 10-fold serial dilutions of your milk sample to arrive at a final dilution of 1:1000. To do so: a. Add 20 µL milk to 180 µL H2O to create a 1:10 dilution in a microcentrifuge tube. (20µL milk in 200 µL final volume is 1:10) b. Mix well by capping the tube and flicking it vigorously. (What happens in the next step if you don’t mix well here?) c.

Make a second 1:10 dilution using 20 µL from the dilution that you just made + 180 µL H2O for 1:100 dilution (

1 1 1 ) as shown in the figure below. × = 10 10 100

d. Repeat dilutions until you get 1:1000 3. Use 10 µL of each dilution in a Bradford assay and observe the color change. Did the cuvette immediately turn bright blue (might be outside the linear range and would not be a good candidate)? If the color matches one of your middle standard curve samples, you are within range. 4. Use this dilution of milk to proceed, and measure it using 5, 10, 20 and 40 µL of the diluted milk. Use Table 3 to record your data and use the blank from Part A. Be sure to wipe the exterior of your cuvette with a Kimwipe.

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Analysis: Drawing a Standard Curve: Now that we have collected the absorbance values of each of the different known BSA solutions (standards) we must plot the standard curve. Generate a scatter plot with concentration on the X axis and absorbance on the Y axis to turn in. A line of best fit must then be put on the data set. Note that this is not the same as a line graph, our line will not connect each of the data points but should best represent the trend at which the data is moving. By using the line of best fit we can determine the concentration of our milk solutions. An important question we must first answer is whether we believe that our standard curve is accurate. Errors in measurement could lead to an incorrect measurement of our milk sample. We can statistically 2 test for the quality of our standard curve by calculating the regression coefficient (R ). This calculation will tell us how closely our data is to a fitted regression line. If the data points for the graph 2 were properly measured we would expect a R value of around 0.98--1.0, any lower would suggest errors in your technique and it is likely that your calculations for the unknown protein concentration milk will be wrong. For a guide on how to perform this graphing in excel please see this video: https://youtu.be/cbtV1Lv4I2k

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Name:_____________Duc Chau_ Section:_______587______

LAB 1: Measurement, Pipette Use, and the Standard Curve Experiment 2 Standard Curve Table 1 Final Protein Concentration

µL of BSA protein stock (0.5 mg/mL) needed

Bradford Reagent

0.15M NaCl for 1.0 mL final volume

Tube

50 µg/mL

0.1 mL

900 µL

0mL

25 µg/mL

0.05mL

900 µL

0.05mL

15 µg/mL

0.03mL

900 µL

0.07mL

10 µg/mL

0.02mL

900 µL

0.08mL

7.5 µg/mL

0.015mL

900 µL

0.085mL

5.0 µg/mL

0.01mL

900 µL

0.09mL

8

7

6

5

4

3

5

2.5 µg/mL

0.005mL

900 µL

0.095mL

0mL

900 µL

0.1 mL

2 0 µg/mL

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Table 2 Final Concentration 50 µg/mL 25 µg/mL 15 µg/mL 10 µg/mL 7.5 µg/mL 5.0 µg/mL 2.5 µg/mL 0 µg/mL

Absorbance 595nm 0.67 0.45 0.21 0.20 0.02 0.01 0.01 0.00

Part B: Protein Concentration in Milk Table 3 My Milk Type = _____6_____ The Dilution of milk I used was: __1:100_________ Tube 1 2 3 4

Unknown Volume 5 µL 10 µL 20 µL 40 µL

Bradford Reagent 900 µL 900 µL 900 µL 900 µL

0.15M NaCl for 1.0 mL final 0.095mL 0.09mL 0.08mL 0.06mL

A595nm

0.13 0.22 0.45 0.65

Concentration Measured by Spec. 14.773 µg/mL 25 µg/mL 51.136 µg/mL 73.864 µg/mL

Concentration of original milk 294.40 µg/µL 250 µg/µL 255.70 µg/µL 184.66 µg/µL

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a. What was your final R value? What does this tell us about the precision of your measurements? My final R2 value is 0.845, it is not an ideal value as this means that my data precision is not that accurate since the ideal R2 value should be close to 1 or around 0.99-0.92. At least the R 2 that I want to get.

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Concetration vs Absorbance.

Concentraition in ug/mL

0.6 0.5 0.4

f(x) = 0.01 x + 0.05 R² = 0.85

0.3 0.2 0.1 0 0

10

20

30

40

50

60

Absorbance at 595 nm Figure 1 Concentration vs Absorbance graph (pre-modified)

b. Look at the final two values of your standard curve on the scatterplot, do they correlate strongly with the rest of your data? Why or why not? Yes, if I were to remove my final two values, it could either help increase or decrease my R 2 value. Ideally, you would want the R2 value to increase but it could also decrease due to number of reasons such as not wiping the cuvette correctly, not doing dilutions correctly, etc. In my case, my R2 value got decreased to 0.0078x.

c.

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What happens to your R values if you exclude the last two values in the curve? Does that change the concentrations of your unknown? If so, which should you use to calculate the unknown concentration in milk? As shown below, my R2 value decreases when I remove the last two values in the curve. Yes, it does change because I get a different slope or equation. Therefore, my concentration changed if the last two values were removed as according to beer’s law, there is an inverse relationship between absorbance and concentration. If my slope decreases, my concentration increases.

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Concentration vs Absorbance 595 nm (modified) 0.6

Concentration

0.5 0.4

f(x) = 0.01 x + 0.09 R² = 0.81

0.3 0.2 0.1 0 0

10

20

30

40

50

60

50

60

Absorbance at 595 nm Figure 2 Modified Concentration vs Absorbance

Concentration vs Absorbance 595 nm 0.6 0.5 0.4

f(x) = 0.01 x + 0.09 R² = 0.81

0.3 0.2 0.1 0 0

10

20

30

40

Figure 3 Modified Concentration vs Absorbance graph

d.

What is the concentration of the milk solutions in the cuvette? Show your work in what you hand in (it’s worth points). Either construct what you did digitally (2+2)/4=1, or take a photo of your work and insert it into the document. The concentration of my milk solutions in the cuvette are 14.773 µg/mL, 25 µg/mL, 51.136 µg/mL, 73.864 µg/mL respectively. I found these values by applying the beer’s law. As we know, we can portray beer’s law into y=mx form because y intercept is assumed to be zero. By plugging in the right units into beer’s law I was able to find my values. For example, x=y/m or x=0.13/0.0088, which would give me a concentration of 14.773 µg/ µL

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e.

f.

What is the concentration of protein in your milk? Did you use all data in calculating the unknown concentration? If you excluded some data, provide a justification. Remember you must account for dilution. Show your work. The concentrations of my protein in my milk were found to be 294.40 µg/µL, 250 µg/µL, 255.70 µg/µL, 184.66 µg/µL respectively. I did use most of my data to calculate the unknown concentrations, most of them comes from table 3. I plugged in my units into my equation c1v1=c2v2. C1 would be the variable that I’m looking for, my v1 is my unknown volume, which is 5 µL, c2 is found from using beer’s law which is 14.773, v2 is always 1mL. (c1)*(5 µL)=(14.773 µg/mL) (1mL). C1 is found to be 294.40 µg/µL after multiplying by my dilution factor of 100.

How does your determined concentration compare to the concentration of protein listed on the milk carton? My determined concentration was very far off as my determined concentration was found to be 246.19 µg/ µL. There are many reasons why there is this much amount of errors. One could be that the dilutions were not correctly done during this lab. Another reason could be that we use Beer’s Law to find concentration that was measured by Spec instead of taken the data directly from Spectrophotometer. All these factors contribute to a high determined concentration compared to the one listed on the milk carton.

g.

Turn in this Data Sheet package (pages 5 and 6) along with a copy of your graphs that you plotted in Excel. Make sure that your graphs display the trend line, the equation for the line, and the R2 values.

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