Arabidopsis Culture Cell and Transformation Arabidopsis thaliana (L.) Columbia ecotype suspension- cultured T87 cells were maintained at 22°C in JPL3 medium with continuous illumination and shaking at 100g. Two-week-old cells were sieved through 500 μm stainless mesh and the remaining filtrate was transferred to a flask containing 20 ml of fresh JPL3 medium for subculture. Transformation of T87 cells was done by culturing the cells in B5 medium supplemented with 1 μM 1-naphthaleneacetic acid (NAA) and 40 g L-1 sucrose. The cells were cultured for one day at 22°C with continuous illumination and shaking at 120g. Next, 10 μL of overnight cultured Agrobacterium transformed with respective vectors were added into the cell suspension and cultured for an additional two days. After co-cultivation, the cell suspension was washed thrice with 10 mL of JPL3 medium supplemented with Carbencilin (250 μg mL-1) by centrifuging at 100g for two minutes. Finally, the cells were resuspended and spread onto JPL3 selection agar plate supplemented with Carbencilin (250 μg mL-1), Kanamycin (30 μg mL-1) an...
The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. “Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin” (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert.
For part one of the experiment, my team asked the question of which cell fraction of the measured pea seedlings will have a higher ratio of chloroplasts? My group tested for the activity of chloroplasts with three different pairs of cell fractions by two conditions of light and dark in three readings. The first two cell fractions, pellet one and two (P1, P2), are the hard sediments found at the bottom of a tube after it has been centrifuged (which are specimen, like the mitochondria and chloroplast, that are isolated from the rest) (Leicht and McAllister, 2016). The last cell fraction used was the supernatant two (S2), which is just the free liquid surrounding the pellet after the centrifuging of P2 (Leicht and McAllister, 2016). To test for this, DCIP (a chloroplast isolation buffer) was used to
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...
Tissue culture protocol of sugarcane consists of five stages: explant inoculation, callus induction, sub-culturing and somatic embryogenic calli production, and regeneration of shoots and rooting. Lastly, acclimatization of the plantlets in the green house.
International Union of Biochemistry and Molecular Biology. [Online].; 2002 [cited 2014 April 21. Available from: onlinelibrary.wiley.com/store.
If a plant cell is places in a hypotonic solution the cell has a lower water concentration to that of the solution. Water will move into the cell by osmosis from a high water concentration outside the cell to a lower water concentration inside the cell through a selectively permeable membrane. The cell becomes turbid
In the case of temperatures the cultures were incubated at each determined temperature. For the UV radiation, cells were exposed to UV light for 10 seconds and then grown in 30oC. For the EtBr treatment, 50ul of EtBr was added to the growth medium and cells were incubated at 30oC. In the case of sunlight exposure, cells were exposed to sunlight directly and grown at room temperature
Plants were transformed with a tfdA gene from Alcaligenes eutrophus (JMP134) using a pCAMBIA1301 plasmid The gene was expressed in the roots. Integration of the gene was confirmed by 2,4-D assay, southern blot and PCR
Have you ever wondered if a plant knew it was about to be dinner? Heidi Appel and Rex Cocroft were perplexed with whether or not plants could communicate with, not only themselves but also other plants, about chemical defenses. According to new research, plants may have their own “cell”-phones. When a hungry caterpillar starts chowing down on a bitter leaf that might just be the case.
The average length of a cell in tap water is 90.0588 micrometers and the average length of a cell in a 10% salt water solution is 75.1838. In comparison the differences between the averages are striking. Demonstrating that the length of the cell shrinks and is undergoing plasmolysis when the salt water solution was introduced to the previously standing cells in tap water. Plasmolysis is the shrinking of the cytoplasm away from the cell wall. Plasmolysis is occurring because when the 10% salt solution is introduced to the elodea leaf the cells in the elodea leaf are submerged in a hypertonic solution. Meaning that there are more solutes outside the cell rather than inside the cell. The solution is hypertonic because the NaCl which composes salt has a high electronic pull. Causing the H2O in the water to attract to the NaCl. When submerged in the salt water solution the elodea leaf cells water is being attracted to the NaCl electric pull so by diffusion the water is pulled out of the
[6 temp] - Darnell, James E., Harvey F. Lodish, and David Baltimore. Molecular cell biology. 4th ed. New York: Scientific American Books :, 1990. Print.
one of the most important staple crops for the world and it now currently feeds
Patil, JG, ML Ahire, KM Nitnaware, S Panda, VP Bhatt, PB Kishor, and TD Nikam. "In Vitro
Ayatollahi, Jamshid, Fatemah Ayatollahi, Ali Mellat Ardekani, Rezvan Bahrololoomi, Jahangir Ayatollahi, Ali Ayatollahi, and Mohammad Bagher Owlia. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 02 July 0005. Web. 23 Jan. 2014. .
Cell culture of plants is an appealing substitute for the whole plant for producing these highly