S-STEM Scholar Anh Nguyen's Blog

  Introduction In lab this week, we did not perform any experiments because we mainly focused on editing and writing our scientific poster about our transformation project with E.coli plasmids and D. caeni  for our conference on 4/13.  The Poster In the poster, we talked about the purpose of this experiment and we discussed about what Deinococcus species was successfully transformed in the past that allowed us to continuing working with E.coli plasmids and the Deinococcus species. In previous experiments, Deinoccocus radiodurans was proven to be successfully transformed with pRAD1 . Below shows the results of the transformed cells on TGY plates with 3 ug/ml chloramphenicol.  These plates show 1 ug of pRAD1.  Below is another result of the transformed cells in a gel performed from colony PCR as confirmation.  Lane 1: 100 BP Ladder Lane 3-5: 861 BP of PCR product Since we have confirmation and evidence that a Deinococcus species have the ability to be transfo...

  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 centrif...

  Introduction Unfortunately since we could not successfully transformed pwtCas9 with D. caeni in Week 13, we will be using existing transformed cells that we have successfully gotten in the other previous experiments to attempt confirmation cells. There are two ways we can perform confirmation; PCR and Plasmid Prep. PCR is essentially the reading of genomic sequences of the cells and since we know that these transformed cells were successfully resistant to tetracycline, we want to see the resistant sequence gene once we load the PCR into a gel. Similarly, plasmid prep allows us to perform plasmid extraction on our transformed cell and loading them into a gel to see if it has the pwtCas9 plasmid within the cells.  Procedure For this confirmation, we used 4 different concentrations of the transformed cells and two controls: - 135 ng pwtCas9  - 550 ng pwtCas9  - 783 ng pwtCas9  - 803 ng pwtCas9  - (+) D. caeni (no antibiotics) - (-) D. caeni (with 2 ug/ml of ...