|A Flower of a Different Color by "Emblazon"||Main project page|
Previous entry Next entry
A Flower of A Different Color
The Promoter group(Binu,Sushma) took the incubated plasmid pSB1A2 with the promoter BBa_R004 ,that we extracted from iGEM 2007 and Spring 2008.But we tried to transform the plasmid from the well kit with electrocompetent cells( Ecoli DH5alpha strain) before centrifugation,hence we lost those sample, due to evaporation. So we decide to go ahead with the plasmid that we got from Spring 2008 kit.We transformed the cells(DH5alpha strain)with plasmid pSB1A2 and pBlue Script and water, the last two were used as control and plated them with ampicillin in the medium,incubated at 37 degrees. We also plated DH5alpha on medium without ampicillin.
The vector group(Angie & Diwash) performed restriction digests of the plasmids psB1A3, psB1A7 & pBluescript. The enzymes used were fast-digest EcoR1 & fast-digest Xbal. We made a master mix for each of these enzymes so that we could just transfer 18 microliters of the master mix to 2 microliters of each plasmid DNA sample. Axel performed a restriction digest on his samples of our plasmid DNA also in order to see if our results would turn out the same. If not, the problems we have been encountering with our gels could be due to pippetting errors or maybe one or more of our stock solutions was made incorrectly. After performing 2 restriction digests on each of our plasmid samples, using a different enzyme each time, we performed electrophoresis of our digested samples, along with undigested samples of our plasmid DNA & Axel's digested samples of our plasmid DNA. Our results were almost exactly the same as Axel's. All of the samples of pSB1A7 were not digested and no bands appeared. The psB1A3 samples that were digested with the EcoR1 enzyme and the Xbal enzyme showed bands between 2,000 and 3,000 base pairs. Some of these psB1A3 samples showed multiple bands, which indicates that the enzyme did not have time to fully digest the DNA. The pBluescript plasmid showed bands around 3,000base pairs, which is what we would expect and served as our control. Since we have been obtaining bad results with the pSB1A7 plasmid we are not going to work any further with these samples. Since we did see some results with the pSB1A3 samples we will continue to work with this plasmid.
Tiffany, Melissa and Rajiv prepared the DNA template for the PCR reaction using the same standards as previously. Some precautions were taken when preparing the DNA sample for PCR, using 2 micro liters of DNA template using two temperatures for the reaction one at 53.7C and at 58C. The sample was stored in Axel's lab labeled with the teams name and date to be tested for amplification over the weekend using electrophoresis. If the samples have no amplification then the amount of the DNA template will have to be increased in order for any amplification to occur. The RNA gel and materials were prepared for testing the RNA during the lab today. The gel and materials will have to be sterilized once more due to contamination by other students in the lab work space that placed unsterilized and RNase free material on the gel. To prevent this from occurring during the RNA preparation we will label all materials to remain in a separate fume hood from other students that are working in the lab on other projects. Tiffany and Melissa will repeat the RNA experiment on Saturday when there is less students around in the lab to limit RNase and other contamination.