840:153g:Projects/project1/2008/11/13

A Flower of a Different Color by "Emblazon"

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Success at Last A Flower of A Different Color Tiffany performed PCR with 1, 2, 4, 8, and 10 microliters of DNA template. The reverse transcription PCR product was left in the thermocycler. Melissa,l Rajiv, and Tiffany analyzed all the PCR products with electrophoresis and 8 out of 10 of the DNA PCR products amplified and the bands were between 2000-2500 bp in length. This could be due to an intron in the gene sequence. Both samples from the reverse transcription PCR showed bands at the expected length (aroung 1500 bp), so this means that it was from DNA contamination in the sample. This conclusion was based on the fact that the second sample was not included in the reverse transcription step. The DNA PCR products were purified and half was sent off to be sequenced. The rest was set aside to be digested with restriction enzymes, purified again, and ligated in to the extracted vector that includes our promoter. The digest will be done by Sushma and Binu on Friday and the ligation with the vector will be done by Tiffany on Monday. On Tuesday the group will all reconvene and transform E. coli with the plasmid. Binu and Sushma double digested the plasmid pSB1A2 each with promoter BBa_R0040,BBa_R0010,BBa_R0051 with spe1 and pst 1.Then we extracted the opened plasmid (pSB1A2) using QIAEGN PCR Purification kit and stored the sample at -20 degrees. Angie & Diwash prepared LB medium by dissolving 25g of LB in 1 liter distilled water,divided it into 2 bottles, added 7.5 g of Agar into each bottle and autoclaved it for about an hour. We then poured plates of LB medium and inoculated 4 of the plates with E.coli. 2 of the plates were inoculated with E.coli containing the plasmid pSB1A3 and 2 of the plates were inoculated with E. coli containing pSB1A3. These samples were received through MIT previously. We will let these plates grow overnight and store them in Axel's lab until Monday, when we will inoculate LB broth with colonies from the plates and grow overnight once again. Then, tuesday we will extract these plasmids using the Gene Jet protocol Axel used previously. We are doing all of this in order to help verify whether or not the samples of pSB1A3 were accidentally mislabeled as pSB1A7. If they were, many of our previous experiments would make much more sense.