Team:Rock Ridge Virginia/Results

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Project Results

Parts

Once we received our parts from IDT we transformed the plasmids in DH5-alpha cells in ampicillin resistant plates.All of our plates (OspA, WSP and GFP) had many colonies due to the fact that we added 3 times the amount of DNA since we had some problems with transformation in our practice rounds. The positive plates were positive and the negative plates were negative.

We picked 5 colonies from each plate and we purified the plasmids using mini-preps. We checked the plasmid purity using agarose gels, all of the colonies had the plamid inside. We did not sent the plasmids for sequencing due to the cost, we just selected one of the five colonies and did a PCR using Gibson Assembly primers. We had a lot of difficulty doing the PCRs because the melting temperature between the primers was not similar. It took us quite some time to get all of amplicons ready, especially WSP to which we had to add b-mercaptoethol to get a good yield.

Our first attempt at PCR

Optimized PCR amplicons :)

For the Gibson Assembly we used a linear ampicillin plasmid backbone (pSB1A3), and the gel purified amplicons for the reaction. Due to the way we designed our primers and parts (composites), we could only do the Gibson Assembly between GFP and WSP. RFP cells are present in the plate and at first we thought it was contamination until we read that it was undigested plasmids.