Restriction mapping of plasmid DNA

Purpose: The purpose of this lab was to characterize a DNA sequence.

Introduction: A plasmid is a small DNA molecule that's separated from a chromosomal DNA and can replicate on its own.  Restriction mapping is obtaining the structural information of DNA by using restriction enzymes. These restriction enzymes cut DNA at specified regions called "sites". 

Methods: We started out by adding the dye into each designated slot of our gel. We were to aim the pipet into the groove without puncturing the gel. If we punctured the gel or placed the gel in the opposite way, this would have ruined our experiment. After adding the dye to each slot, we let it sit for a day so the restriction enzymes could cut at their specified region. The next day, we came back and measured how far each DNA region traveled down the gel. 

Data

Graphs and charts

Discussion 

    Our results from this lab came out to be very close to what they should have been.  The only aspect of our results that did not come out ideally is the number of cuts that were made in the final two columns.  While there should have been three distinct cuts in the fifth column, and four in the sixth.  Our gels only showed two cuts in the fifth column and three in the sixth.  A potential reason for this outcome may be that we simply could not see the cuts due to a lack of pigment in the gel.  This pigmentation issue was probably derived from the beginning of the experiment when the liquids were being placed in the gels.  If any amount of the contents from the reaction tubes escaped during the piper ting process, it would make the fragments more difficult to detect.  This means that when loading the last two wells, a large amount of the liquid escaped, and therefore we could not see the extra fragments.  In the future, we would be more careful and precise when transferring the fluid from the reaction tubes to the wells. When labeling our fragments based on size, we knew that the larger fragments didn't travel very far, versus the smaller fragments, which went the largest distance.  With this in mind, we made general estimations on our own gel.  In doing this, it is possible that the lengths we assigned to our fragments may be off.  However, when considering the data, it is all compared to each other, so there should not be much wrong with what information we came out with in the end.  All in all, this was a successful experiment.

Conclusion

To conclude our lab, restriction map information is important for many processes used to manipulate DNA. In this lab for example, one application was to cut a large strand of DNA into smaller pieces to allow it to be sequenced. We were successfully able to characterize a DNA sequence through this process. 

Gel map artfully drawn out by Mr.Filipek (super artistic and well-done)

Emma adding the DNA loading dye to our gel (super successful)

Our whole classes gels all together in the electrophoresis chamber (most of them look amazing good job class)

Our lab group's gel after the electrophoresis was complete 

pGLO Transformation Lab

Hello and welcome to our fabulous P.Glo lab done by Aliya, Emma, Cara, and Ally (your fav senior ladies who value and appreciate you Mr.Filipek) 

Purpose

The purpose of this experiment was to perform and prove genetic transformation which is insertion of a gene into an organism in order to change it's trait. In this experiment we were testing the insertion of the GFP(green fluorescent protein) gene into the P.Glo plasmid of the E.coli cells. 

Introduction

In this experiment, E.coli is a bacteria that is commonly found inside of mammals and birds and is used to breakdown food into small molecules that can be transferred into your blood. It is a very basic prokaryotic cell and only lives a short time. Most types of E.coli are good for you but there are a very few select types that can be poisonous to humans such as E.coli found in raw meat. Genetic transformation, in this experiment is used to transfer new DNA from the GFP to plasmid of the E.coli cells. A plasmid is a small circular piece of DNA where the GFP can be inserted in order to show new traits within the cell. The insertion of GFP into the pGlo makes the cell resistant to antibiotics.

Methods

We started of by using two micro test tubes one of +pGlo (containing pGlo) and one of -pGlo (not containing pGlo). We then placed 250 microliters of CaCl2 into each tube. Next the two tubes we're placed on ice. After this, we put a sample of the E.coli bacteria into each tube using a loop. Immediately, we inserted pGlo plasmid DNA into the positive pGlo tube but not in the negative pGlo tube. Then we incubated (let them sit on ice) for 10 minutes. Right after this, we performed a heat shock on both tubes. Next, we put 250 microliters of LB nutrient broth in both tubes. We then placed 100 microliters of the solution from each tubes onto 2 Agar plates each, resultin in 4 total plates. One plate of +PGlo LB/amp, one of +pGlo LB/amp/ara, one of -pGlo LB/amp, and one of -pGlo LB.

Data

Graphs and charts

Drawn-out representations of our Agar plates and their contents after the lab was completed

Discussion

Our results from this lab were a direct result of the proccess of genetic transformation. Some of the dishes expressed the pGLO trait while others did not because of this proccess. Those that did express the trait those that expressed the "glow in the dark" trait were those with pGLO and LB/amp. The plate with LB/amp/ara contained more  colonies because of the sugar that was added to the plate that helped with growth. If we were to make adjustments to this lab, we would put more of each substance of each plate in order to see more colonies then what we did the first time. It's difficult to tell whether our results are completely accurate due to how little of data that we have. As predicted, the petri dishes that had the GFP inserted into the plasmid glowed in the dark, while the petri dishes without the plasmid did not. The insertion of the GFP into the plasmid then makes it resistant to antibiotic. The LB plate has no growth because it has no protein to resist antibiotic. 

Conclusion 

Through this lab we were able to prove that pGLO causes the bacteria to be able to resist the antibiotic. 

References 

http://scienceforkids.kidipede.com/biology/cells/ecoli.htm

Pre-lab selfie (super excited)

Receiving the translation solution (super fun)

Icing the solutions (super chill) 

Getting the E.coli (super thrilling)

E.COLI (yay)

Heat killing the solutions (super amazing)

Aliya and Emma in the action (feat. Tape and coffee)

Petri dish! (Super scientific)

Mixing! (Super cool and fun)

All the Petri dishes in their final stage of the lab! ( super exciting and biological) 

+pGlo LB/amp/ara

-pGlo LB

+pGlo LB/amp

-pGlo LB/amp

How To: Extract DNA From a Strawberry

Purpose: Hello! This was one of our favorite labs because we all love strawberries a whole lot. The purpose of this experiment was to see if there was a way to extract a strand of DNA from one whole fresh strawberry. 

Introduction: DNA is found in all living organisms and in this lab our goal was to extract a strand/fibers of DNA from the strawberry. 

Methods: First we mashed up the strawberry in a ziplock bag in order to make the DNA more extractable. Then we mixed the mashed up strawberries with the extraction buffer (the soapy water mix). After letting the mixture soak through a coffee filter into a graduated cylinder, we collected the liquid after a few minutes. We then added a few drops of ice cold alcohol to the strawberry and soapy water solution. We made sure to let the alcohol drip down the side of the graduated cylinder instead of dropping it straight in. After a minute or two a white cloudy layer appeared. This was the DNA! After stirring the pipet in the mixture, the DNA climbers could be pulled out and examined.  

Discussion: Our results from this lab successfully proved this lab true. DNA could be successfully extracted from a strawberry as long as we followed the correct steps, which we eventually did. The only thing that went wrong was our first attempt at the lab. We first mixed the mashed up strawberry with the ice cold alcohol to find nothing so we knew then that we had to approach the lab differently. Eventually we figured it out and successfully extracted the DNA from the strawberry. If we were to improve our tactics and techniques in this experiment, we would have not first mixed the alcohol with the strawberry solution. The results of our experiment proved that DNA fibers/strands are found in strawberries. 

Conclusion: To conclude this experiment, DNA can be extracted from raw strawberries as long as it is mixed with an extraction buffer, filtered through a coffee filter and mixed with alcohol. Our results from our lab support this conclusion. 

References: 

DNA Extraction From Straberries (Schoology) 

Pictures

The mashed up strawberries!!!

Adding the alcohol to the solution!!!

Extracting the DNA (feat. Mr. Filipek) !!!

Cell Communication Lab

Purpose

The purpose of this experiment was to observe cell communication between two strains of yeast (A-type and Alpha-type), both of the two being opposite sexes.

Introduction

Cell Communication is essential for organisms in order for signals to turn into responses within the cell. In this lab we will be studying methods by which yeast cells partake in cell communication. It is our job to determine what type of cell signaling  these yeast cells use and how these cells send signals to each other. We will complete this lab with the use of yeast, Petri dishes, and microscopes. 

Methods

We began this lab by separating samples of the a-type, the alpha-type, and a mixture of the two in order to determine the reproduction rate and how the yeasts go about it asexually as well as sexually. We placed each of the wet yeasts in its own Petri dishes and let them sit over night. Before we wrapped up the lab on the first day we measured the yeasts under a microscope. We put broth in the test tubes and placed them into an incubator overnight,. The next day we scraped the dried yeast off the petri dishes and mixed if with water and put each yeast on a glass slide and put it under a microscope. We then measured our data to find the change in yeast cells over time. 

Mixed Yeast (Day 1)

A Type Yeast (Day 1)

Alpha Type Yeast (Day 1)

Alpha-Type Dry (Day 2)

Mixed Dry (Day 2)

A-type (Day 2)

Mixed Wet (Day 2)

Data

Mixed Yeast Culture Data

Alpha-Type Yeast Culture Data

A-Type Yeast Culture Data Table

Graphs and Charts

Graph of the percent change of yeast cells over time (haploid and diploid)

Discussion

In the Cell Communication Lab we measured the difference between the different yeasts (alpha type, A-type, and mixed) to see their sexual/asexual production. We observed that there was not much difference between the a-type and the alpha-type strains of yeast because they are both able to reproduce asexually, there was not much difference in budding of the cells. But, when the alpha-type and A-type were mixed together it is observed that right away there is already diploid zygotes and diploids zygote budding from the cells beginning to grow towards each other because they are opposite sexes. This goes to prove that though yeasts are able to reproduce asexually, bringing the two opposite sexes together causes cellular communication to occur by the sending and receiving of signals to reproduce at a more rapid rate. One thing that could be done differently in this experiment was counting of the different haploid and budding haploid cells. More test trials would have been more efficient but we spent a majority of time trying to get pictures to accurately represent our data. Another thing would be to use different settings on the microscope to get a closer look at the cells and be able to examine the mixture culture closer. 

Conclusion

In this experiment we see that the most favorable condition for yeast reproduction is when yeasts are mixed with a-type strains and alpha-type strains. This is because of the cell's ability to to communicate through signaling between different sexes and as long as the environment is favorable, diploid zygotes are formed and divide.

References

All I Want For Christmas Is Mitosis

Gettin' into the Holiday spirit!

Plant Pigmentation and Photosynthesis Lab

4A Plant Pigment Chromatography

Purpose

The purpose of this experiment was to use Chromatography paper to identify plant pigments in spinach cells. We were testing to see if certain types of pigments were in spinach and the solubility of these pigments (Rf values). The relationship between the pigment and the solvent is the Rf value.

Introduction

The different pigments plants contain are carotene, xanthophyll, chlorophyll a and chlorophyll b. Paper Chromatography is used to separate the molecules because of the varying solubility of molecules in a certain solvent. It does this by separating the pigments using capillary action. This happens because solvent molecules are attracted to the paper as well as each other. When the solvent moves up the paper, it carries the pigments with it.  The distance the pigments travel is dependent on their solubility.  

Methods

In this lab we began by cutting a strip of chromatography paper into a pencil shape. (A long strip with a pointed end). Next, we scraped spinach cells off of a spinach leaf with a coin and dragged them across the chromatography paper, making sure that this green line did not go past the pointed end of the chromatography paper. We then hooked the chromatography paper onto a hook on the bottom of a stopper that was to go in a 50-ml graduated cylinder. The graduated cylinder had 1 cm of solvent in the bottom of it. We then made sure that the solvent only touched the pointed end and did not emerge the strip portion. The rest of the lab we let this chromatography paper sit and waited to record our results when the period ended, after the chromatography paper had diffused all of the spinach's pigments up the paper. 

Rubbing spinach cells onto chromatography paper

Placing the paper in into the test tube that holds the solution

Waiting..

Waiting...

Still waiting....

Measuring the distances traveled!

Measuring the distance of each band number 6.3-.6

Data

Graphs and Charts

Discussion

Our results contained the Rf values for 4 specific pigments found in spinach leaves: Carotene, Xanthophyll, Chlorophyll a, and Chlorophyll b. These results were directly related to how far each of them traveled up the chromatography paper. Each pigment was able to travel different distances up the paper because each pigment had different solubility levels and the chromatography paper is able to show this. A trend found in our data was the relationship between the distance of each pigment traveled and the Rf values of each of the same pigments. As a result if this, a conclusion can be made that the further the distance a pigment travels up the chromatography paper, the higher it's solubility is. The ones who traveled the furthest had the lowest Rf values and vice versa. These results were accurate and support our answer. If we were to change anything or re-do certain aspects of this lab we would have correctly measured the pigment's distances during the actual lab. Instead we just took pictures of the distances and referred to them because we were running out of tine by the end of this lab. Other than this, our lab was successful. Our results support this conclusion.

Conclusion

To conclude this experiment, spinach contained these 4 pigments: Carotene, Xanthophyll, Chlorophyll a, and Chlorophyll b. The solubility of these pigments were a directly related to how far the pigment traveled. Chlorophyll b had the lowest Rf value and traveled the shortest distance while Xanthophyll had the highest Rf value and traveled the furthest. Our data correctly supports this answer. 

References

http://www.gcisd-k12.org/cms/lib4/TX01000829/Centricity/Domain/783/2014%20Paper%20Chromatography%20of%20a%20Spinach%20Leaf%20Lab.pptx

4B Photosynthesis/ The Light Reaction

Purpose

The purpose of this experiment was to test whether light and chloroplasts are required in order for light reaction to occur.

Introduction

Many factors can either inhibit, help, or have no effect on Photosynthesis (light reactions). This lab was conducted to study this. DPIP, a compound posing as an electron acceptor, water, a phosphate buffer, and a spectrophotometer were used in this lab to reach the answer to this question. A dye-reduction technique is used in this experiment to study photosynthesis. 

Methods

To begin in this lab, we added the correct amount of DPIP, distilled H20, and phosphate buffer to each of the 5 cuvettes. The only one that did not contain DPIP was the first one. We made sure to wrap cuvette 2 in tin foil because no light was supposed to enter this cuvette. Each of the 5 cuvettes were measured in the spectrometer right after their mixtures were created. After each of these results were recorded, a 5, 10, and 15 minute time period was measured for each cuvette again. While one cuvette was in the spectrometer, the other 4 (except for number 2) were placed in front of a heat sink filled with water that was placed in front of a light.The heat sink's purpose was to absorb the heat so the cuvettes would experience no heat. After each of the 5, 10, and 15 minutes of the cuvettes were finished, we recorded our results.  

Measuring DPIP

Filling a cuvette with DPIP

Still filling

Exposing the cuvettes to light

All of the materials used

Unboiled chloroplasts 

Data

                                    % Transmittance

Graphs and Charts

Discussion

Unboiled/dark and Unboiled/light both had a significant increase in transmittance within the first five minutes of the experiment which shows that photosynthesis occurs in both light and dark environments while the chloroplasts are still alive. Contrarily, the chloroplasts that were boiled had very little growth as time went on which showed that the denatured chloroplasts hardly undergo photosynthesis.  The change in transmittance that was apparent with the boiled chloroplasts may be due to some of the sample not being fully denatured.  If this experiment were to be done again, it would be beneficial to make sure all of the chloroplasts are fully denatured so that there would be even less photosynthesis occurring within the boiled chloroplasts.  At the same time, it may be inevitable to have a portion of the sample could survive being boiled.  This comparison also shows that the light has much less of an impact on photosynthesis occurring than the nature of the chloroplasts, as those which were simply not exposed to the light still transformed, while the boiled chloroplasts were incapable of undergoing photosynthesis. The results we obtained are in sync with what we thought would happen because in order for plants to go through photosynthesis there needs to be a presence of light (in order to start the light/dark reactions) and chloroplasts, in order to absorb the light. If we conducted the experiment again, in order to see a more significant transmittance change, we would need to be more careful with how we mix or cuvettes as well as being more persistence with how much of each substance is being tested. Also, testing each cuvette between a certain time period rather than so close together so the time we collect our data is more exact, because that affects the transmittance measured.

Conclusion

To conclude this experiment, light does not have as big of an impact on photosynthesis as chloroplasts do. Our data generally supports this theory except for the slight differences in the transmittance levels of the boiled chloroplasts. In order for our data to completely support our theory, all of these levels should have been exactly the same. 

References

http://www.nature.com/scitable/content/the-light-and-dark-reactions-in-the-14705803