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Questions and Answers
In spectrophotometry, what is the primary purpose of using a blank solution?
In spectrophotometry, what is the primary purpose of using a blank solution?
- To introduce color to colorless solutions.
- To dilute the protein sample for better readings.
- To increase the absorbance of the sample.
- To calibrate the spectrophotometer and eliminate absorbance from non-protein components. (correct)
Why is it important to multiply by the dilution factor when determining protein concentration from a Bradford assay standard curve?
Why is it important to multiply by the dilution factor when determining protein concentration from a Bradford assay standard curve?
- To adjust the concentration back to the original, undiluted sample concentration. (correct)
- To account for the volume of Coomassie Blue added.
- To correct for any errors in the spectrophotometer readings
- To normalize the data across different protein samples.
A researcher observes that a protein sample in Coomassie Blue shows a low absorbance value, close to that of the blank. What is the most likely explanation?
A researcher observes that a protein sample in Coomassie Blue shows a low absorbance value, close to that of the blank. What is the most likely explanation?
- The spectrophotometer is malfunctioning and needs recalibration.
- The protein concentration in the sample is very high.
- The protein is not binding to the Coomassie Blue dye effectively. (correct)
- The cuvette was inserted incorrectly, blocking the light path.
In an experiment, a researcher predicts that increasing the concentration of a substrate will increase the rate of an enzyme-catalyzed reaction. Which of the following correctly identifies the hypothesis and prediction?
In an experiment, a researcher predicts that increasing the concentration of a substrate will increase the rate of an enzyme-catalyzed reaction. Which of the following correctly identifies the hypothesis and prediction?
A scientist is studying the effect of temperature on bacterial growth. They maintain constant humidity, light, and nutrient levels in all experimental groups, while varying the temperature. Which variable is the independent variable?
A scientist is studying the effect of temperature on bacterial growth. They maintain constant humidity, light, and nutrient levels in all experimental groups, while varying the temperature. Which variable is the independent variable?
A researcher is testing a new drug to treat high blood pressure. One group receives the drug, and another group receives a placebo. What purpose does the placebo group serve in this experiment?
A researcher is testing a new drug to treat high blood pressure. One group receives the drug, and another group receives a placebo. What purpose does the placebo group serve in this experiment?
Why is reproducibility considered a crucial aspect of experimental design?
Why is reproducibility considered a crucial aspect of experimental design?
Given a standard curve equation of $y = 2.5x + 0.1$, where 'y' represents absorbance and 'x' represents concentration, what is the concentration of a protein sample with an absorbance of 0.6, considering a dilution factor of 5 and a volume of 1 ml?
Given a standard curve equation of $y = 2.5x + 0.1$, where 'y' represents absorbance and 'x' represents concentration, what is the concentration of a protein sample with an absorbance of 0.6, considering a dilution factor of 5 and a volume of 1 ml?
Why is it important to avoid withdrawing liquid too quickly when using a micropipette?
Why is it important to avoid withdrawing liquid too quickly when using a micropipette?
A researcher needs to accurately measure and dispense 35 μL of a solution. Which micropipette would be the most appropriate to use?
A researcher needs to accurately measure and dispense 35 μL of a solution. Which micropipette would be the most appropriate to use?
What does the 'first stop' on a micropipette plunger primarily control?
What does the 'first stop' on a micropipette plunger primarily control?
In a serial dilution, if the final desired concentration is 0.5 M and the initial concentration is 10 M, what is the dilution factor?
In a serial dilution, if the final desired concentration is 0.5 M and the initial concentration is 10 M, what is the dilution factor?
Why are serial dilutions performed when working with micropipettes?
Why are serial dilutions performed when working with micropipettes?
What is the relationship between absorbance and protein concentration in a Bradford assay?
What is the relationship between absorbance and protein concentration in a Bradford assay?
A scientist is performing a serial dilution. They start with a 5 M stock solution and need to create 5 mL of a 0.2 M solution. What volume of the stock solution is required?
A scientist is performing a serial dilution. They start with a 5 M stock solution and need to create 5 mL of a 0.2 M solution. What volume of the stock solution is required?
A series of measurements are taken with a micropipette, yielding volumes of 9.8 μL, 9.9 μL, and 10.1 μL, when attempting to pipette 10 μL. Which is true about the micropipette's performance?
A series of measurements are taken with a micropipette, yielding volumes of 9.8 μL, 9.9 μL, and 10.1 μL, when attempting to pipette 10 μL. Which is true about the micropipette's performance?
Flashcards
Blank (Spectrophotometry)
Blank (Spectrophotometry)
Used to calibrate the spectrophotometer to ensure absorbance readings reflect only the protein, not the solvent.
Coomassie Blue
Coomassie Blue
A dye (Coomassie Blue) that binds to proteins, changing color from brown to blue, measured at 595 nm.
BSA (Bovine Serum Albumin)
BSA (Bovine Serum Albumin)
A protein with known concentration, used to create a standard curve for protein quantification.
Standard Curve
Standard Curve
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Hypothesis
Hypothesis
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Prediction
Prediction
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Positive Control
Positive Control
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Negative Control
Negative Control
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Micropipette Volume Ranges
Micropipette Volume Ranges
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How Micropipettes Work
How Micropipettes Work
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Micropipette Stops
Micropipette Stops
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Accuracy
Accuracy
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Precision
Precision
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mL to μL Conversion
mL to μL Conversion
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Serial Dilution
Serial Dilution
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Bradford Assay & Absorbance
Bradford Assay & Absorbance
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Study Notes
- There are three different micropipette types, each with a different use
Micropipettes
- P10 (Red) has a volume range of 0.5-10 microliters
- P100 (Yellow) has a volume range of 10-100 microliters
- P1000 (Blue) has a volume range of 100-1000 microliters
- The line on P100 and P10 represents a decimal to allow more accurate volume measurements
- Micropipettes work due to air displacement
- The first stop takes up the desired volume
- The second stop expels the desired volume
- The accuracy range of a micropipette is less than 3% of the intended value when used correctly
- Accuracy decreases as you approach the lowest value of that micropipette's range
- For example, to upvote 10ul, use a P10 (red), not a P100 (yellow)
Accuracy vs Precision
- Accuracy is how correct a value is
- Precision refers to how close values are to each other
- Precision produces reproducible results and is expressed as the standard deviation in a set of data
- There are 1000 microliters per 1ml
- When micropipetting, do not withdraw liquid too quickly
- Slowly allow the plunger to return to its position with your thumb and do not let it snap back
- Dispense liquids against the sides of the tube to eject the full volume
Bradford Assay
- Bradford Assay is used to determine protein concentration
Serial Dilution
- Dilutions are performed when solutions need small amounts of solvent, which allows for more accurate dilutions
- First, find the dilution factor (final concentration / initial concentration)
- Then, use the c1v1=c2v2 equation and solve for whatever variable you need
- Solve for v1 to get the amount of solute, then subtract the amount from the total volume to get the amount of solvent
- Spectrophotometer determines protein concentration which measures solutions that contain the protein to figure out the concentration using the absorbance of that solution
- As absorbance increases, the protein concentration also increases
- A blank is used to calibrate the spectrophotometer and ensure that the absorbance is only due to the protein
- The solution must have some indicating color to use the spectrophotometer and requires Coomassie Blue
- Coomassie blue is a dye that changes from brown to blue when proteins bind to it
- It is recorded at a wavelength of 595 nm
- Compare a solution to a solution of a known protein (BSA - Bovine Serum Albumin) after finding the absorbance
- A=-log (T) is the change from transmittance to absorbance
- When loading a cuvette avoid the clear sides and make sure that the arrow of the cuvette is oriented in the direction of the light path
Standard Curve Interpretation
- A standard curve uses linear regression where absorbance is along the y axis and concentration is along the x axis
- It should help predict absorbance when concentration is known or concentration when absorbance is known
Getting Concentration from Bradford Assay
- Plot the standard curve and get the equation
- Knowing the absorbance, solve for x
- Make sure the x is multiplied by the dilution factor, then divide by the volume
Experimental Design
- Hypothesis: A proposed explanation for a phenomenon based on observations and knowledge
- Prediction: If x occurs then y will occur in a scenario where x and y are either independent or dependent variables
Types of Variables
- Independent: Variable changed by the researcher
- Dependent: Variable being recorded
- Controlled: Variable of the experiment that does not change
Controls
- Positive Control: Groups where the conditions received guarantee a positive result
- Negative Control: Groups where the conditions received guarantee a negative result
- Reproducibility is important in experiments
- Controlled experiments should have constant variables
- Another scientist in a completely different lab should be able to replicate experiments and reach the same results
Model Organisms
- Examples: C. elegans, Drosophila flies, Mice, Yeast, Zebrafish, Xenopus, Arabidopsis
- Easy to breed and maintain
- Have short generation time
- Large number of offspring
- Well studied genome
- Easily manipulated
- Not as complex
- Efficient manipulation of genome
- Less ethical concerning than humans
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Description
Learn about micropipettes and their accuracy. There are three main types of micropipettes: P10, P100, and P1000, each with a different volume range. Accuracy decreases as you approach the lowest value of that micropipette's range.
