pGLO Transformation Lab

Purpose

The pGLO transformation lab was designed to transfer genes from different species into the bacteria E. Coli. We needed to insert two genes into the E. Coli, a gene resistant to ampicillin and a jellyfish fish gene that causes it to glow under UV light. We also counted how many colonies in each of the cultures and whether they showed the pGLO gene. There were four cultures, two with the pGLO gene and two without. The two with the pGLO gene were labeled LB/amp and LB/amp/ara, the two without the pGLO gene were labeled LB/amp and LB.

Introduction

This lab is all about gene transformation and cells ability to assimilate foreign DNA. Cells have a natural ability to accept genes as their own but to a degree. Genetic transformation is used in many areas of biotechnology. This ability to create new more successful organisms strongly supports the use of biotechnology. The lab was designed to show how effective biotechnology is can be in present day science. Some cells such as eukaryotes have more layers which make it harder for some genes to get through. However some cells such as prokaryotes have few layers such as E. Coli so their assimilation of foreign DNA is much easier. The goal of this lab is to get bacteria to grow, be ampicilin resistant and have it glow in the dark. To makes this happen, it was necessary to know how move genes from one organism to another. This only happens through the use of plasmids. Through the use of plasmids you are able to add genes that you desire and have the plasmid act as a medium or a method of transportig that gene. 

Method

In the lab we took the plasmid or the pGLO gene and put it in the +pGLO test tube, and didn't put any plasmid into the -pGLO tube. We then took the tubes put them on a rack and put them in a cup of ice for ten minutes after that we put E. Coli into the test tubes and gave the tubes a quick heat shock for fifty seconds and then left the tubes with the plasmid and E. Coli to incubate for ten minutes at room temperature before we added liquid broth or LB to all the cultures to help fuel cell division by giving them food to use. We took all the agar plates with the various combinations of the pGLO gene and ampicillin resistant gene and E. Coli and taped them upside down (to prevent condensation) and put them overnight into a 37*C incubator. 

Addition of E. Coli to the petri dishes.                             Tubes chilling on ice before being added.

           
                                           Our lab group's undesired results.

                                         Desired results from a different lab group.


Discussion
This pGLO transformation lab gave us unpredictable and highly undesired results. The intended goal was to have E. Coli grow and integrate the pGLO gene so it would glow under UV light. The data and results were not what we expecting even though we followed the instruction, however for such skewed results to occur there must've been a human error somewhere along the lab. Comparing our four plates we only had growth on one dish. The bacteria was a white opaque color. When we tested the E. Coli to see if it transformed accepting the pGLO by using the UV light we had no glow meaning the pGLO gene unsuccessfully integrated into the E. Coli. Looking over our data, results, and the conversation we had with our teacher we concluded that we did not add enough plasmid to get the E. Coli to transform. Finding out what our mistake was a critical component of realizing ow important certain steps were such as cooling the tubes and keeping them o the ice. The chilling kept the DNA closed, preventing it from mutating. Heating the tubes for fifty seconds was very crucial because it gave the plasmid an opportunity to enter the DNA when it was denatured and be assimilated into the E. Coli. Adding and following the directions precisely is really important for the success of a lab because if it doesn't work out your lab's results can be invalidated.

Conclusion
The pGLO transformation lab was a definite and obvious failure for our lab group as it did not glow under UV light as was the intended goal. Although we had disappointing results where our bacteria did not glow we did however have a minor success due to the fact we had some E. Coli growth. We followed the instructions correctly, but we have come to the conclusion that due to the fact we did not dd enough plasmid nor pGLO gene, E. Coli did not have anything to transform or assimilate as it's own DNA.

Reference
"PGLO Transformation Lab." Flashcards. N.p., n.d. Web. 17 Mar. 2015.

"PGLO Transformation Lab." YouTube. YouTube, n.d. Web. 17 Mar. 2015.

"PGLO." Wikipedia. Wikimedia Foundation, n.d. Web. 17 Mar. 2015.

Restriction Mapping

Introduction: The goal of this lab to see and compare the DNA sequence strand to three others. The original strand will be placed next to the other strands and compare and see what sites are close and related. We used three restriction enzymes Pst1, Hpa1, and Ssp1. These were the different restriction enzyme digesters. Then after setting up the DNA it is key and essential to store it in the electrophoresis chamber over night to get the results and look over. 

Purpose: The purpose of the restriction mapping of plasmid lab is to know that a restriction map of a piece of DNA is like a fragment of the DNA. The importance of knowing the number of cuts sites present in DNA sequence for each restriction enzyme, but the needed positions of those cut sites relative to another. These steps make it easier to determine similar and unfamiliar DNA sequence strands. 

Methods: 

In this lab, we had 3 different restriction enzymes in different combinations, and 5 identical DNA samples. We were provided with 5 different tubes that contained the combinations. We then took very small pipettes and placed the samples into the gell's wells. These are the specific combinations of what was placed in each well.

1. Marker Lane

2. BLANK

3. DNA & Pst1
4. DNA & Pst1/Hpa1
5. DNA & Pst1/Ssp1
6. DNA & Pst1/Hpa1/Ssp1

Once the mixtures were placed in the wells, we put the gel tray into the electrophoresis chamber. Once in the chamber, an electricity is run through all of the gels to move the DNA through the gel. The smaller pieces moved farther than the bigger pieces that stay closer to the wells. After the process was finished, we were able to put our gel on a "light-box" so we could see the bands and measure the distance of the pieces movement and compared them to the marker lane. Pictures of the gel on the light box are below.

Data:

This picture shows the distances of the marker lane as they compare to the other lanes. 

Discussion: The restriction mapping of plasmid DNA gave us near perfect results. We had a variation of strands and cutting sites. When looking at our gel tray on the light box, you can see the relationship between the sites. For an example the last strand has the same distance of cutting site. It is difficult to see the multiply cutting sites on the fourth row. This shows how the relationship between each strand of DNA differs for each other. We consider the consistencies of our trends to be very similar. No one strand is far different. Yes, there will be different cut sites, but nothing was drastically abnormal. The results from this lab were great. Anyone looking at the results could tell the correlation between each one. The results came as we predicted and how they should actually look. Again the whole of Lab Group 2 views this restriction mapping of plasmid DNA lab as a complete success, and as one of our better resulting labs. 

Conclusion: The restriction mapping of plasmid lab was a success. As the results were close to perfect, we got everything we thought we would see, and it had great variation. The lab was so successful due to the fact that we followed the instructions to the T.  When we examined the stained gel over the light it made the cut sites clear as possible. We consider this lab VERY successful. 

Reference: 

● "Circular Restriction Mapping." Circular Restriction Mapping.N.p., n.d. Web. 18 Mar. 2015. 

● "Restriction Map." Wikipedia.Wikimedia Foundation, n.d. Web. 18 Mar. 2015. 

● "Restriction Mapping." Restriction Mapping.N.p., n.d. Web. 18 Mar. 2015.