840:153g:Projects/project1/2008/10/02

A Flower of a Different Color by "Emblazon"

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A flower of a different color Today the RNA extraction was completed by Tiffany, Rajiv and Melissa producing a total of 1ml RNA in -70C storage using the TRIzol instructions [1]. The manual is listed under Trizol pdf. The primers for the Biobrick and the Sequence are completed and offically ordered by Axel. The primer sequence does have some problems associated with it, since it is likely to have primer dimers (4 base pairs matched). And there is a chance of shorter fragments formed around 1381 base pairs, the gene length we would prefer is 1532 base pairs long. According to the Amplify 1.2 computer program the primers should work well and actually amplify the gene. Next week we will test the primers that were ordered and test the RNA and DNA extractions to see if the gene is present in the sequence. The electrophoresis Axel performed showed just about the same results as did the two Angie and Diwash performed previously. We have come to the conclusion that there is definitely something wrong with either the E. coli cultures that were used to extract the plasmids from, or the plasmids could've been kicked out of the DNA and not present in the E. coli at all. Today we, Angie, Diwash, Binu and Sushma, decided to attempt at extracting the same plasmids (psB1A3 and psB1A7) from the Registry to see if we could achieve any better results. We extracted each part, psB1A3 and psB1A7 from both the Spring 2008 parts kit using the protocol on [[2]] and the iGEM 2007 parts kit using the protocol on [[3]]. We are planning on trying to isolate these 2 plasmids using 2 different sources: the registry and Axel's stock of E. coli (strain DH5alpha). We are also going to isolate a "backup" plasmid, bluescript, in case we are not successful with isolating psB1A3 or psB1A7. Today we also peformed the procedure for bacterial transformation using the protocol from [[4]]. We performed 6 transformations: 2 for psB1A3, 2 for psB1A7, and two for our controls. We will be using the plasmid bluescript as a control to ensure we do have competent cells and water on LB medium to ensure the cells we are working with are not dead. The final steps which includes the inoculation of the strains onto the ampicillin containing medium was completed by Diwash and Axel. We hope to see cultures growing on the plates on Friday. Monday, Angie and Binu are going to grow new stocks of E. coli (DH5alpha strain)containing the plasmids psB1A3 and psB1A7 and also grow stocks of E. coli containing the plasmid bluescript. Tuesday we plan on beginning to isolate these 3 plasmids using the protocol on [[5]]. Then we will do a restriction digest of the plasmids and check for the correct number of base pairs by doing electrophoresis.