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...
While the previous experiment identified colonies containing recombinant DNA, the patching experiment distinguished which colonies contained the hlyA fragment and which ones did not. Colonies that could cause haemolysis of the blood agar plate indicated that recombinant DNA taken up contained the hlyA fragment ligated with pBluescript, which is the desired subcloning product. The hlyA fragment contains the hlyA gene which encodes for a haemolytic protein that causes the red blood cells in the blood agar to lyse. Therefore, non-haemolytic colonies were transformed with pBluescript plasmid ligated with the pK184 fragment and were not able to cause haemolysis as no hlyA gene was present. In theory, this experiment allowed for the aim to be achieved as it identified colonies with the desired product. Inoculating certain colonies in broth culture allowed for gel electrophoresis to be carried out and confirm if the aim of the experiment has been
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...
The germinating seeds consumed almost no oxygen throughout the experiment in the 10-degree C water bath. I think that this is because when an organism cools down, all of its cellular functions slow down.
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 results for the various conditions differed dramatically. As seen in the table, “Data Collected During Time Interval” the reference test tube remained at a trasmittance level of 100% for all five experimental tests. The control solution remained fairly constant for all five tests, but did vary slightly after the five minute time interval.
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
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.
Patil, JG, ML Ahire, KM Nitnaware, S Panda, VP Bhatt, PB Kishor, and TD Nikam. "In Vitro
[6 temp] - Darnell, James E., Harvey F. Lodish, and David Baltimore. Molecular cell biology. 4th ed. New York: Scientific American Books :, 1990. Print.
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.
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. .
one of the most important staple crops for the world and it now currently feeds
Cell culture of plants is an appealing substitute for the whole plant for producing these highly