840:153g:Projects/project1/2008/12/04

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A Flower of A Different Color

Today Diwash and Angie performed a restriction digest of our extracted pSB1A2 plasmid DNA samples using a fast-digest enzyme, EcoRI. We did this to verify whether or not our gene, cytochrome p450 was inserted into the plasmids which already containing 1 of 3 different promoters. If the gene did insert, we should see bands of DNA at approximately 4,000 base pairs.(pSB1A3 is 2,079 bp, the promoter BBa_R0051 is 49 b.p, BBa_R0010 is 200 bp, BBa_R0040 is 54 bp, and our gene is about 2,000 bp including introns) We had 24 samples we were working with, so we made enough "master mix" of 10x fast-digest buffer, EcoRI enzyme, and distilled water. We then transferred 2 microliters of plasmid DNA from each sample into tubes containing 18 microliers of master mix, and let sit in a 37 degree C water bath for 30 mins. We then transferred 4 microliters 6x loading dye into each sample tube and transferred the whole sample now containing 24 microliters into the lanes of the agarose gel previously set up by Sushma and Binu. We used a 100bp molecular weight marker for a standard to compare our bands of linear plasmid DNA with. We ran electrophoresis of our digested plasmid DNA samples for 30 mins at 150V. We then took a picture, which showed no bands of DNA, or no results. This could be due to the fact that the overnight cultures of e. coli containing the plasmid DNA in tubes of LB broth were left in the refrigerator for almost a week after growing overnight at 37C. This probably gave the cells in the cultures time to die, resulting in the destruction of the plasmid DNA, making it impossible for us to extract. Since we have used the same extraction procedure & restriction digest procedure before, but with overnight cultures grown at 37C and extracted the next day with good results, we can conclude that the storage of the overnight cultures in the refrigerator for ~ a week is most likely the problem.

Sushma, Rajiv and Binu digested and purified the PCR products [1] and did the gel electrophoresis of the same at 150 V for 30 minutes.

Melissa and Tiffany figured out from the results the reasoning why the gene sequence split into two fragments was due to a possible restriction site within the middle of the gene that was not initially found when the gene was analyzed for restriction sites. The other possible reason for the gene to split is due to the possible chance of an intron within the sequence.