Final Lab Practical, Lab Practical 1

A and pure water

Which cells (figure 2) have undergone plasmolysis? What could be added to these cells to reverse plasmolysis?

- bag could've come loose - not being consistent in time - not drying the bag before weighing

Why are there differences among the data sets between you and your classmates? Would you expect the standard error for the cumulative change measurements to increase or decrease over the course of the experiment? What do your standard error calculations in the table in question #3 indicate?

It calibrates the spectrometer. Gets rid of the impurities before measuring other tube's. Blank has everything you're not measuring like ONPG.

Why is it important to compare all your samples to the "blank tube" when taking your absorbance measurements?

Only one variable can be tested at a time. The experimental variable is enzyme concentration, therefore substrate concentration must be kept high enough so that the rate of the reaction is independent of the substrate concentration. In other words all enzymes must be saturated for the results to be valid.

Why was it important to use an extremely high concentration of ONPG for this experiment? (Figure 6)

A plant cell with an osmolarity of .8M will undergo plasmolysis if placed in

a solution greater than .8M

In lab 5, you will be looking at cells from

an onion root tip

The GFP gene is located near specific regulatory sequences on the plasmid that normally function to turn on and off arabinose metabolism genes. This regulatory sequence is switched on by the presence of the ARA-C protein, which is produced by the araC gene located on the plasmid. What would you need to add to the growth media in order to turn on the GFP gene?

arabinose

Consider the table #10. Which of the following best accounts for the result in the 20 minute tube?

between 15-20 minutes the enzyme is not operating at a maximum rate because most of the substrate has been used up

Given two molecules of the same molecular weight and shape, the one with greater _______ will migrate through the agarose faster.

charge

During Metaphase

chromosomes line up on the metaphase plate

What technique will you be using to separate dye molecules?

gel electrophoresis

Look at the equation for calculating osmotic pressure. If the temperature is held constant but the total solute concentration increases, the osmotic pressure of the solution would ___________.

increase

Examine the data table #5. These results support the hypothesis that the rate of osmosis

increases with concentration

Two dye molecules equal in size and charge may have different migration rates through an agarose gel because of their difference in...?

shape

Plasmids are

small circular DNA that are found in bacteria

We will measure the amount of ONP produced by using a

spectrophotometer

In lab 1 you examined cross sections of ______ and _____ to observe the diversity of cell types found in multicellular organisms.

syringa leaves/animal intestine

To calculate the cumulative change in weight for each bag, you subtract the weight of the bag at each time point from its weight at _____________. (see page 20 question 2.)

t = 0, the starting time point

We will be examining the effects of time, concentration, and ________ on enzyme activity.

temperature

During Anaphase

the chromosomes begin to separate

3000mg/60min/30cm2 = 1.67mg/min/cm2

Calculate the rate of osmosis (mg/min/cm^2) for the 10% bag.

yes, lactase is constantly breaking down ONPG without being used up.

Enzymes are said to act as catalysts of reactions because they act to speed up the chemical reaction but are not used ip in the reaction. Does your data support this statement? Explain.

What stage of the cell cycle is this? M

Metaphase

Lactase would be completely denatured. The extreme temperature increased the spontaneous breakdown of ONPG into ONP. It is non-enzymatic.

Most students found ONP production in tubes incubated at 100 degrees C. Does lactase function at this temperature? Explain.

You will be measuring the intensity of the yellow color in the reaction mixture after adding lactase. This yellow color is produced by

ONP

Why does an enzyme act specifically on a particular reactant or class of reactants?

Only certain reactants can fit into the active site of the enzyme

What stage of the cell cycle is this? P

Prophase

The osmosis rate would be quicker..

Suppose that a 20% solution of NaCl had been used instead of sucrose. How would the results differ from those obtained with 20% sucrose? Why? Assume the bag is also impermeable to NaCl.

What stage of the cell cycle is this? T

Telophase

Mitotic Index

# of cells in mitosis/total # of cells

For the results shown #19, 10ul of .8ug/ul plasmid solution was added to 250ul CaCl2 and after heat shock 250ul of LB broth was added. 100ul of this mixture was plated. Calculate the transformational efficiency (in transformants/ug).

(10ul)(.8ug/ul) = 8ug 8ug/510ul = .016ug/ul ( .016ug/ul)(100ul) = 1.6ug 34 transformants/1.6 ug = 21 transformants/ug

Students observing prepared slides of root rips found 20 cells in mitosis out of a total of 100 counted. The mitotic index for these cells is

.20

Examine the table on page 40. Which molecule will have the slowest migration rate?

1 (750g/mol, -8, squiggly)

Examine the table on the bottom of page 40. Which molecule will have the fastest migration rate?

3 (200 g/mol, -6)

Calculate the transformational efficiency in transformants/cells plated.

34/2000 = .017 transformants/cell plated = 1.7 %

How long is interphase for these cells if mitosis is 4 hours?

4/0.2= 20--> 20-4= 16 hours

How long is metaphase in fish embryos if 4 out of 28 mitotic cells were in metaphase and mitosis takes 6 hours?

4/28(6hrs)= 0.86 hours

What is the osmotic pressure of a 2mol/liter solution of sucrose? osmotic pressure=CRT R=0.082 (liter atm)/(K mol) T= 293 K

48 atm

Consider the data table #5. What is the cumulative weight gain for the 30% bag after 20 min?

7.9 g

What stage of the cell cycle is this? A

Anaphase

Why don't the cells in plate A fluoresce?

Arabinose is required to turn on the GFP gene and it is not present in the growth media.

Examine the results of the DNA analysis for an STR for 5 individuals in the figure #18. If Mom(A) has a child (C). which of the 3 other individuals (B, D, E) could be C's father? How could we use STR's to determine definitely which man is C's father?

D & E could be C's father, but B could not. We cannot tell by looking at one STR, since many people may have the same number of repeats for one particular STR. We would have to look at multiple STRs in order to tell which man was C's father.

37º body temperature

Each enzyme has a characteristic temperature at which it operates most efficiently. This is known as the optimal temperature for that enzyme. What is the optimal temperature for lactase? Does this make sense based on what you know about the natural environment of the enzyme?

A. The molecular weight of NaCl is 58g/mol and therefore a 10% NaCl solution has a much higher molar concentration that a 10% sucrose solution. In addition, NaCl dissociates into Na+ and Cl- ions.

Examine figure 1. The dialysis bag is shown in the photo contains a 10% sucrose solution and the beaker contains a 10% NaCl solution. Which of the graphs shows the expected results?

A

Examine figure 2. Which photo (a or b) shows cells in a hypertonic solution?

b. The area of the 20% bag was much greater than the area of the 30% bag.

Examine figure 3. Which of the following best accounts for the surprising difference in the results between the 20 and 30% bags? a. The osmotic pressure (CRT) in the 20% bag was greater. b. The area of the 20% bag was much greater than the area of the 30% bag. c. There was a leak in the 20% bag d. The rate of water leaving the 20% bag exceeded the rate of water entering. e. none of the above.

The first step in the Osmosis lab procedure is to:

Fill the beaker with distilled water and soak the empty dialysis tubing

40º and 60º decrease enzyme activity. The enzyme is denaturing. The structure of the protein is changing therefore the enzyme cannot catalyze.

For which two of the temperatures tested is enzyme activity the lowest? Does this make sense based on what you know about enzyme action and protein structure?

At 100º the enzyme is destroyed. The heat breaks the bonds.

Given your knowledge of protein structure you should be surprised by the results obtained for the tube in the boiling water bath. How can you explain this result?

Successful transformation requires that the bacteria be heat shocked. Why is this necessary?

Heat shock provides the stress needed to get the bacteria to take up materials from their surroundings.

When in hypertonic solution cytoplasm/protoplasm separates from cell wall and that's how you know plasmolysis is occurring.

How did you determine the osmolarity of reed onion cells? Your explanation should be a discussion of the biological phenomena, not a simple restatement of the data.

Water will move out of the 10% bag. In the 20% bag there would be no net movement because the solution is isotonic. In the 30% bag the water will move in the bag.

How would you expect your observation to change if the beaker contained 20% sucrose instead of water? How would the curves of cumulative change vs. time for each concentration change?

ONP would accumulate in their blank and therefore their readings would be inaccurately low.

In preparation of their blank a pair of students accidentally combined the enzyme and substrate before adding NaCO3. How would this mistake affect their subsequent absorbance reading?

If a dialysis tube is filled with a 20% sucrose solution and placed in pure water it will

Increase in volume

What stage of the cell cycle is this?

Interphase

Remain constant

The rate of the reaction is measured by the slope (rise/run) of the curve. Compare the rate between 0-10 minutes with the rate between 10-20 minutes. Does it increase, decrease, or remain constant?

Over time there will be no net movement. Water is going in the same rate it is going out.

What would you expect the 30% bag to weigh when t= 2 hours? What forces eventually cause the curve to level off?

The solution surrounding a cell is isotonic (equal concentrations on either side of the membrane). What will happen?

There will be no net gain or loss of water across the membrane

Why is ampicillin added to plates A and B?

To kill all cells that haven't been transformed.

Electrophoresis of the contents of tube E resulted in four bands. Which of the following explains this observation?

Tube E contains a mixture of 4 different dye molecules.

When independent variables are discrete (no values fall between them). For example to illustrate the relationship between cell length and species.

Under what circumstances is a bar graph appropriate?

When you want to illustrate how each part fits into the whole. In other words when you want to indicate percents.

Under what circumstances is a pie chart appropriate?

Increase in concentration, increase in rate of osmosis.

What is the relationship between rate and concentration?

Turnover time

duration of mitosis/mitotic index

What is the correct order in preparing the blank for the enzyme lab?

enzyme --> Na2CO3 --> ONPG

What is the function of the bla gene on the plasmid?

it gives the bacteria carrying the plasmid resistance to ampicillin

What is the function of the ori sequence on the plasmid?

it triggers DNA replication of the plasmid

In the human digestive tract, lactase catalyzes the breakdown of

lactose

Lactase catalyzes the conversion of

lactose to glucose and galactose

The migration rate of DNA fragments is directly related to their....?

length

Plasmolysis is when a cell

loses water and shrinks

During Prophase

the nuclear membrane disappears

The Mitotic Index is

the number of cells in mitosis divided by the total number of cells

Genetic transformation is

transferring a gene into an organism