S-STEM Scholar Micheal Dockham's Blog

  Monday 10.28.24     Prepped cultures for the week. Making 6 total.           2x D. Ficus WT - 20ml          2x D. Ficus (transformed) + CMR - 20ml          2x E. coli + prad1 + AMP - 20ml Discussed plan for tomorrow and rest of the week. The goal is to perform plasmid extractions on the 6 cultures made today. We are testing WT and Transformed Ficus against both primers to confirm the presence of pRAD1 in our transformed d. ficus. We will be comparing this to how the primers behave with the actually pRAD1 extraction as well.  The goal overall is to finally confirm the presence of our pRAD1 vector in our transformed d. ficus cells. This has proven to be challenging. Both for ficus and for deinococcus in general. . Tuesday 10.29.24 Both WT and "Transformed" ficus cells didn't appear grown enough, so they were left to incubate for another 24 hours.  In the meantime, a Zyppy Plasmid Ext...

 Monday 10.21.24 With current complications in PCR reaction results. We have decided to run PCR on our wild-type strain of D. ficus, using both cmr+ and amp primers to verify if contamination of species, both transformed and wild-type strain has happened.  Plates made on Friday were observed, with wild-type strain having normal growth, while our transformed strain grew on one plate somewhat well, and was left in the incubator to grow at temperature of 37C. Primers for PCR were located and the proper request emails were sent out.  Along with this transformation confirmation attempts we have decided to also explore D. ficus ability to survive and possibly thrive in higher pH environments. Apparently and according to research ficus should be able to grow in high alkalinity environments up to a pH of 10. We want to explore and confirm this, make observations and try and understand how these strain can survive in such a high alkaline environment.  We have decided to use m...

 With the electroporation machine shock pod down, and the funds lacking to replace it we changed our focus to chemical transformation. The goal, similar to the electroporation goal, is to insert the pRAD1 vector into D. Ficus via chemical transformation Here we attempt what I am calling the Tuohy Transformation protocol, a method described by Dr. Tuohy that uses simple chemical transformation methods to perform the procedure.  This involves incubating our cells in R2B media for 24 hours at 30C, this should have been 37C. Cells were pelleted and resuspended in fresh R2B(100ul) 40uL of CaCl2 in added and mixed This is then incubated for 15 minutes on ice. With delays in the lab, such as lines to the stations, the cells sat for around 45 minutes in ice 8ul of pRAD1(368ng) elution was added to 30ul of culture mix This was then incubated at 37C for 90 minutes. 1.5ml of R2B was not added tho, as the protocol called for. instead more cells were added. This was then placed back on ice...

 Began performing "carnage test" on aliquoted D. ficus culutres stored in the -80C Trest was to find optimal settings with the machine, based on our theory that the more "carnage" the higher the likelihood of transformation success.      40ul from -80C were thawed and placed in the cuvette for electroporation.          Settings were:               3000V               25uF              Infinite resistance, which uses then natural resistance of the cells versus a dialed in resistance.               Cells were then diluted and plated, placed in the 37C for 24 hours and observed the next day               No "carnage" was observed and plate had full growth. Later in week performed experiment again on 2 samples with different settings, to observe any ...

 Semester begins.  Week was mostly spent making media and prepping for upcoming projects. Monday 8.12 Made media for the week     200ml TGYB     250ml TGYA Tuesday 8.13     Inoculated D. Ficus plates from freeze back Wednesday 8.14     OD values were taken, however values were too low and culture was left to grow.     Started prepping for Glycerol Washing. Friday 8.16     Performed glycerol wash on roughly 400ml of  D.ficus culture for future electroporation experiments     Created 40ul aliquots as well as many 100ul+ aliquots as well