Week of October 28th, 2024

 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 Extraction was performed on the e. coli (pRAD1) cultures, where we extracted 2x 30ul eloutions containing; 110ng/ul and the later containing 76ng/ul. These were then confirmed for the correct plasmid via a EP 1% gel. This confirmed the vector had been properly extracted, be it less then desired quantities extracted. In the future, I've been advised to not pack the pellet for extraction to tightly, especially with such high OD levels, both being over 3.0. 

Wednesday 10.30.24

           Experimented with making buffers, only to learn the way I was going about it was the incorrect way. Sat down with Dr. Tuohy and revaluated my approach. 

            "transformed" cells have proven to be difficult to grow in broth, with WT sometimes being difficult in standard 3:1 TGY All flask inoculated and left to incubate failed. Any plates made this week worked well and grew. 

Thursday 10.31.24

            Made test culture for pH experiment with d. ficus. Sodium Carbonate + Bicarbonate(12mM)

            pH slightly increased in alkalinity after autoclave. (.34) 

            Let to sterilize in autoclave.

            Tested standard 3:1:1 TGY for base/control pH which came to a pH of 7.23            

Friday 11.01.24

            


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