840:153g:Projects/project1/2008/10/28

A Flower of a Different Color by "Emblazon"

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Entry title A Flower of A Different Color Vector group (Angie and Diwash), did the restriction digest of the plasmids pSB1A3.1 and pSB1A3.2 samples isolated from MIT and pBLUE isolated from Dr. Axel laboratory by the use of two restriction enzymes: fast digest Xbal and SpeI. We use only the two plasmids because they showed proper result of plasmid band around 2000bp in the previous experiment. As, we need both the restriction sites such as Xbal and SpeI in order to insert the target DNA. Hence, we did the restriction digest by using both the fast digest enzymes. We run the the samples in the agarose gel electrophoresis along with marker and pBLUE as a control, but the result was very different from the previous laboratory work. The plasmid bands look very bright and even don't show the appripirate band sizes. Also, the band sizes are variable from 2000 to 2400 base pair. Simply, the reslut of same plasmid sample digested in same manner in the privious experiment with same restriction enzyme variate from each other. Sushma and Binu worked on figuring out which promoters to use as they didn't observe any culture growth with plasmid pSB1A2(with the desired promoter BBa_R0040). But the controls (pBluescript, DH5alpha and water) worked well. We came to the conclusion that it must because the plasmid, pSB1A2, was not present in the registry even though according to the registry the part is available in the Source Plate 1000, 4C as given in [1]. Hence we decided to try out the available and working promoters that could be grown in DH5alpha. We took out the following plasmids - BBa_R0051 [2],BBa_I14032 [3],BBa_R0011 [4],BBa_R0010 [5]and BBa_R0040[6] from iGEM 2007 and Spring 2008 kit. We stored these plasmids at - 20 degrees to do the transformation with DH5a Ecoli cultures the next lab. Rajiv, Tiffany and Melissa worke on DNA extraction of the rest of the frozen leaf material. DNA extraction was left off at prepcitating out the DNA in isopropanol and to be completed and tested on Thrusday with the PCR product that was produced Saturday using the last of the DNA stock. Possible reasons for problems with the PCR product could have been due to the actual concentration amount in the Diluted and poor pipetting techniques. Also due to thawing out the product and refreezing it which breaks down the tissue samples and causes damage to DNA and RNA. To prevent overly thawing out the product we will keep the tissue product frozen at all times when working with by constantly adding Liquid Nitrogen to sample. We could have also damaged our DNA sample by repeatedly thawing and freezing it, so we will store it at 4C in the refrigerator.