The Cell Cycle: Mitosis

Background

Mitosis, also known as karyokinesis, is the process by which the nucleus divides, resulting in two diploid daughter cells. It is the M phase of the cell cycle, which also includes the G1, S, and G2 phases. In G1, the cell grows and performs normal functions. In S, DNA is replicated. In G2, the cell prepares to undergo mitosis. G1, S, and G2 are referred to as interphase-this is where the cell spends the vast majority of its lifespan.

Hypothesis

If observed with the compound microscope, the onion root tip slide should show that the majority of cells are in interphase.

Materials

-Compound microscope
-Onion root tip slide
-Whitefish blastula slide

Methods

We worked in groups of two. After focusing the microscope with the high power objective, we took turns calling out the amount of cells in each stage and recording our numbers (these stages are interphase, prophase, metaphase, anaphase, and telophase). We then calculated and graphed the percentage of cells in each phase.

Statistical test

Consider that it takes 24 hours for onion root tip cells to complete the cell cycle. You can calculate the amount of time spent in each phase of the cell cycle from the percentage of cells in that stage. 

Percentage of cells in stage x 1440 minutes = ____ minutes of cell cycle spent in stage

Dependent Variable

The dependent variable was the number of cells in each stage of the cycle in each field of view.

Independent Variable

The independent variable was the field of view observed.

Confounding Variable

We kept the brightness constant throughout the experiment, we made sure that all fields of view were fully occupied every time we counted, and the microscope was undisturbed besides adjustments to focus.

Replication & Sample Size

In the four fields of view we observed, we counted 297 total cells. 

Control

There was no control.

Data

The table below shows how many cells were found in each stage in each field of view, the overall percentage of cells made up by each stage, and the amount of time spent in each phase over a 24 hour period. 

Data Analysis

The pie chart displays the information in the table found above in graphical format.

As predicted, the majority of the time was spent in interphase.

Conclusion

The hypothesis was supported by our data, as almost three quarters of the cells were in interphase. Unsurprisingly, prophase-the second longest stage- occupied the second largest portion of the chart. This experiment could act as a basis for comparison to other experiments. These onion cells were in arrest; thus, we could perform an experiment using onion cells that have been exposed to a growth hormone and compare the percentages of cells in each of the stages. 

Literature Cited

The "AP Biology Lab #3: Mitosis" packet we were provided was used.

Transformation Lab Writeup

Transforming E. Coli With GFP and Ampicillin Resistance

Introduction

Background

Through the process of transformation, bacteria are capable of taking up DNA from their environment (in this case, through exposure to a plasmid containing the desired genes). This gives the bacteria new genetic information that is stable and can be passed down to future generations. In this experiment, we transformed  E. Coli with pFluoroGreen, which contains genes for ampicillin resistance and Green Fluorescent Protein (GFP). Furthermore, we exposed some of the cells to IPTG, which is required for GFP to be expressed. Thus, the experiment demonstrates differential gene expression.

Hypothesis

If E. Coli is exposed to both IGTP and pFluoro-Green, then fluorescent bacteria with resistance to ampicillin will grow.

Materials and methods

Materials

• BactoBeads E. Coli GFP Host
• pFluoroGreen plasmid DNA
• Ampicillin
• IPTG
• CaCl2
• Growth Additive
• ReadyPour Agar
• Recovery Broth
• Petri plates (small and large)
• Plastic tipped micropipettes
• Toothpicks (sterile)
• Inoculating loops (sterile)
• Microcentrifuge tubes
• Automatic micropipettes and tips
• 37 degree C & 42 degree C water baths
• Thermometers
• Incubation Oven (37 degrees C)
• Ice
• Markers
• Bunsen burner/hot plate
• Gloves
• Long wave UV light

Methods

We began by labeling our + and - microcentrifuge tubes. Then, we transferred CaCl2 solution into the - tube, and then added about 15 colonies of E. Coli to the same tube. We vortexed the solution, and noticed that it was still very clear. So, we added more colonies and vortexed again. It was still clear, but less so; thus, we continued by extracting half of the 500 microliter solution and placing it in the + tube. Afterwards, we added pFluoroGreen to the + tube, while leaving the - tube alone. We iced both tubes for 10 minutes, placed them in a 42 degree C bath for 90 seconds, and then iced them for 2 more minutes. We then added 250 microliters of recover broth to each tube, and then incubated them for half an hour in a 37 degree C bath. While we waited, we labeled our four agar plates as follows: -DNA,-DNA/+Amp,+DNA/+Amp,+DNA/+Amp/+IPTG. After recovery ended, we transferred the solutions to their appropriate plates and spread the cells over the plates using the inoculating loops. After 5 minutes, we stacked and taped our plates together, and incubated them overnight. Finally, we placed them under UV light in the morning and observed our results.

For specific steps refer to: http://www.edvotek.com/site/pdf/223.pdf

Statistical Methods

We are going to calculate transformation efficiency and then plot the class’ data on a graph, establishing standard deviations.

Results

Dependent variable: Number of E. Coli colonies present and whether or not they were fluorescent/ampicillin resistant

Independent Variable: Exposure to the plasmid and/or IPTG

Confounding Variables: All E. Coli extracted from same plate, all plates incubated for same amount of time, all measurements that needed to be equal in both samples kept equal (example: both had 250 microliters of recovery broth added), sterility maintained

Replication/Sample Size: We did not repeat our trials, but we will be using the class’ data, so we have over 20 samples.

Controls: The -DNA and -DNA/-Amp plates served as controls

Data: Measurements still have to be taken; none of our bacteria expressed fluorescence

Data Analysis: Measurements must be taken and data must be shared in the class before we can analyze data. While none of the bacteria expressed fluorescence, we did have some colonies grow-albeit few.

Conclusions

None of our bacteria displayed fluorescence. At first, I thought it might have been that they simply required more time to incubate. However, even after inspecting the plates under the UV light again after some time, we had no glowing colonies. Much of the class lacked glowing bacteria too; in fact, I only saw one plate with glowing bacteria. Perhaps the E. Coli simply failed to integrate the DNA from the plasmids. Human error is also a likely cause-in our case, I think we might have transferred the colonies from the plate to the tube improperly. If we had the opportunity to repeat this experiment, I would make sure to follow the instructions more carefully and pay special attention to the initial transfer of the E. Coli into the microcentrifuge tube. Adding more colonies might have helped our chances of having some of the bacteria express fluorescence, too.

Literature Cited