Ligation of EGFP Insert to Plasmid Vector
Ligating the EGFP cDNA into a pET41a (+) plasmid in order to create recombinant expression plasmids and run these ligations through gel electrophoresis to visualize the DNA and check the success of the ligations. Five ligation reactions were generated, two actual ligations and three controls, with a total final volume of 20uL each. NcoI and NotI are restriction endonucleases whose purpose are to reduce non-recombinant plasmids from forming and to prevent undesired rearrangements during ligation.
Ligation one was a 1:1 molar ratio pET-41a (+) vector: egfp insert that used 50ng NotI/NcoI cut pET-41a (+) DNA, 7ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert made up of 50ng NcoI/NotI cut pET-41a (+), 21ng egfp insert DNA, 1uL of DNA ligase, and the proper quantity of water to dilute 10x ligase buffer to a 1x final concentration. Water was sterilized and deionized. The remaining three ligation samples served as controls. Ligation three contained 57ng uncut pET-41a (+)/EGFP recombinant plasmid DNA and sterile water. Ligation 4 was a negative control that consisted of only sterile water. Ligation five lacks DNA ligase but has the same properties of the 1:3 molar ratio pET-41a (+)/EGFP vector.
The given DNA ladder sample and each individual ligation samples were run on 40ml of 0.8% agarose in 1x TAE buffer for approximately sixty minutes at 110V. The appropriate volume of 6x GelRed track dye was used after it was diluted to a final concentration of 1x and incubated for thirty minutes. Finally, the gel was illuminated under UV light and analyzed.
Tra...
... middle of paper ...
...It allowed access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analysis. First, the pEGFP-N1 plasmid nucleotide sequence was found by using the NCBI nucleotide database program. SnapGene viewer illustrated the restriction enzyme cut sites used to cut EGFP gene from the pEGFP-N1 source plasmid. Then the pET-41a (+) vector sequence was found by using the AddGene Vector Database. A new DNA file representing the recombinant pET-41a (+)-EGFP plasmid was built by virtually cloning the EGFP gene insert into the pET-41a (+) vector sequence. The plasmid was virtually cut utilizing the pAD1 sense primer and pAD1 anti primer from the PCR procedure. A restriction digest experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cut the PCR product at least once was HgaI and XspI.
Data table 1 Well plate Contents Glucose concentration A 3 drops 5% sucrose + 3 drops distilled water Negative B 3 drops milk+3 drops distilled water Negative C 3 drops 5% sucrose +3 drops lactase Negative D 3 drops milk +3 drops lactase 15+ E 3 drops 20% glucose +3 drops distilled water 110 ++ Questions B. In this exercise, five reactions were performed. Of those reactions, two were negative controls and one was a positive control.
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Figure 2 shows the results of the electrophoresis. Lanes 5 and 7 indicate the fragments obtained when the plasmids are digested with both restriction enzymes, indicating the approximate fragment size for the hlyA gene, the pK184 plasmid and the pBluescript plasmid. This is useful for identifying the recombinant DNA needed for this experiment
Recombinant DNA technology: Sub cloning of cDNA molecule CIH-1 into plasmid vector pUC19, transformation of XLI-Blue Ecoli & restriction mapping.
Takahashi, Y., et al. “Analysis of Promoter binding by the E2F and pRB Families In Vivo: Distinct E2F Proteins Mediate Activation and Repression.” Genes 14 (2000): 804-816.
Even though the American colonies developed original American beliefs through westward expansion; British Enlightenment ideas and Anglicization provided the foundation for American ideals, proving the culture that emerged in the mid-18th century colonies to be indistinct from Britain.
Lila is a second grade student who participated in a Primary Spelling Inventory and the reflection of her results are as follows. After her spelling inventory was finalized I noted that the student spelled ten of the twenty-six words correctly giving her a power score of 10/26. Most of the words that she mastered was in the Late emergent and early of Letter Name Alphabetic stage. I also noted that Lila accomplished 36 features out of 56 total features during her spelling inventory. Based on the results of the Primary Spelling Inventory the orthographic features that Lila recognizes are the consonants, short vowel, blends, and is familiar with diagraphs. Although she mastered blends which falls in the late Letter Name-Alphabetic stage she failed to master diagraphs which is the middle stage.
Surgical errors are seen in every hospital; however, hospitals are not required to report such incidents. Unintended retained foreign objects, often abbreviated as URFOs, are among those events that are often not reported.
The two modes of analysis that will be used to identify an unknown insert piece of DNA would be plating the transformation cells onto LA plates that have either ampicillin or chloramphenicol and PCR. We will use the PCR thermocycler to denature the restriction enzymes that were specifically used to assimilate the vector DNA. It is important to use the PCR thermocycler because denaturation of the restriction enzyme will prevent the restriction enzyme from cutting the vector DNA, after the insert DNA has assimilated to the vector DNA. After the addition of specific primers that complement the base pair to its corresponding target strand, PCR will be used. Subsequently, Taq polymerase will be used to determine whether the insert DNA has been properly assimilated to the vector DNA. Within this specific situation, the target strand will be the insert DNA. After we let the PCR thermocycler run for approximately 2 ½ hours, we will then put our PCR products in the gel and run the gel to completion. After the gel has run to completion, we will then take a photograph of the gel using the UV transilluminator with the assistance of our TA. If the insert DNA was properly assimilated to the vector DNA, then our corresponding gel photo would have one band. After the cells have been transformed, we would g...
“Molecular and Cellular Cloning - Boundless Open Textbook." Boundless. Boundless, n.d. Web. aaaaa 22 May 2014.
Artemia franciscana, known commonly as Brine shrimp, are aquatic arthropods within the animal kingdom who can readily adapt to extreme conditions. While they mainly live in saltwater lakes, such as the Great Salt Lake and the Caspian Sea, Artemia are able to survive in most inland saltwater excluding ocean (Gonzalo and Beardmore, 2012). Their main source of nutrition comes from Phytoplankton and microalgae: organisms that require sunlight to prosper. In addition, Artemia reproduces both sexually and through parthenogenesis, processes that require specific abiotic conditions for temperature and salinity*. In fact, Wear et al. (1986) states that higher temperatures are more effective in reproduction and maturation of Artemia. Primarily, this experiment is important for acknowledging the ecology of Artemia as well as their biodiversity in a time of climate change. Understanding habitat requirements is essential in aiding preservation, survival and reproduction.
In this experiment we examined myosin, tropomyosin and troponin, and the effects different temperatures have on the shortening of each muscle protein. A muscle is a bundle of fibrous tissue found in a human or animal body that has the ability to contract. This contraction produces movement in and maintaining the position of body parts. Temperature is directly associated with the effectiveness of muscles movement, with an increased temperature there is also an increased rate of fatigue (temperature at normal).
Pigs are complex creatures with an interior structure that is similar to the human body. The purpose of the pig dissection was to recognize the numerous organs and organ structures in the pig's body in comparison to the human body. In spite of the fact that the pig fetus was bigger than what was normal, the lab went well and all the important parts of the pig were present. Yet the discoloration of the organs was unbalanced from what was expected. The whole pig was one beige shading with what looked like blue bruises. This could be due to the measure of time the pig was preserved for. Additionally, the heart was observed to be smaller than expected. It was covered by the lungs and was appeared to be compact because of the steady pumping of blood.
Step 4:Make sure the person holds the clothespin between their thumb and index finger and squeeze until the two ends meet.
The objectives of this lab were fulfilled. During the dissection, diagrams of the rat were conducted from several different perspectives including both the dorsal and ventral views. Throughout the dissection, the prior knowledge that was theoretically learned in class, assisted in allowing the group to have a more complete and thorough understanding of the different organs, and parts that were present in the rat. This helped reveal the several different similarities and differences in the circulatory, digestive, and respiratory system when comparing the rat and human.