Week 16 (4/22-4/26): Regrowing our transformed cells and PCR Gel

 Introduction 

In Week 16, we grew broth culture and performed PCR on our transformed cells which we loaded onto a gel this week. The issue we had about our culture was that there was little to no growth from our transformed cells in antibiotics. This time we will regrow them again in broth but also plates as it may give us different results due to different underlying factors. 


Results

Below is our PCR Gel. 

This was loaded with a 100 BP ladder. In each wells contains 5 ul of our 783 ng PCR solution with 2 ul of dye. From there picture shown, there is not much of the genetic appearance. We believe that using only 5 ul of PCR solution wasn't sufficient enough and will plan to use 10 ul of it in the future. 















For regrowing our transformed cells, we passaged 135ng pwtCas9, 550ng pwtCas9, 783ng pwtCas9, and 803ng pwtCas9 and control D. caeni plates onto new TGY plates with the same antibiotic concentration. At the same time, we also grew them into new centrifuge tubes with the same amount of mixture as last week. 

Unfortunately, after 3-4 days, there was again no growth in our centrifuge tubes except for positive D. caeni and no growth on our plates except for positive D. caeni. 


Discussion
We concluded that our transformed cells are dead cells as they cannot grow at all in antibiotics. This means that we cannot continue confirmation with these cells as they are now invalid. We will not be passaging these cells into freezebacks but will have to restart the transformation process again. 



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