840:153g:Projects/project2/2008/11/11

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The Sweet Smell of.....E.coli?

Today Matt and Jared performed gel electrophoresis on the PCR product that they had created last thursday of part BBa_J45120 using the forward primer that we ordered from IDT and the newest reverse primer that we ordered from IDT in an attempt to make our final product biobrick compatible. The double digestion and the purification of the double digestion of part BBa-I765001 that Surabhi, Diveena, and Aaron performed last week was also loaded on the same gel. The results of the gel electrophoresis suggested that the double digestion of BBa_I765001 was successful as was the purification of that double digestion. This was indicated by the fact that there was a band in each respective lane that was slightly smaller than 3000 bp and the double digested product should be 2971 bp. The results of the elecrophoresis of the PCR product was a smear in each lane with no intense bands indicating that nothing was amplified. The pBlue control lanes contained intense bands at the top suggesting that the problem with the PCR was not due to the protocol but due to something else. Based on the results that we have, we determined that the problem with the PCR is due to problems with the new reverse primer that we designed as we had amplification of BBa_J45120 with the original forward and reverse primers.

To resolve this problem we propose to try 3 things. First, because the annealing portion of our new reverse primer is 8 bp shorter than that of our first reverse primer, we performed PCR amplification again but with 3 temperatures below 48 degrees C which was the lowest temperature tried originally. We also used our originally designed forward and reverse primers and proceed normally with our project using this non-biobrick compatible part. Finally, performed a double digest of part BBa_J45119 which contains our wintergreen part that already has the TetR promoter removed. Proceeding in this way will result in a final product that is biobrick compatible. We will proceed with our experiment with the first of these three that produces positive results, i.e. we have confirmation using gel electrophoresis that we have DNA corresponding with the size of the wintergreen part that we need to ligate to the end of our UV promoter.

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