Week of October 21st, 2004

 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 multiple buffers to a range of pH adjusted media. This includes citrate, phosphate, TRIS and calcium-bicarbonate as our buffers. This will allow us to have a pH range from 5 to 10 in increments of 1, in both broth and plate form. 

I wonder if these conditions will possible "convince" D. ficus to produce a bio-file, something we have seen a number of other deinococcus species exhibit. 

Tuesday 10.22.24

Cultures that were created before leaving Monday were thrown out due to a miscommunication error. 

Still needing to perform PCR reaction, we used plates that were grown on Friday as that was the best option for us to continue and not lose motivation. 

4 PCR reactions were completed. 

Each reaction contained 23ul of master mix solution, including primers, and 2ul of lysed cell and/or pRAD1 extraction. (73ng/ul)

They were then set into PCR machine and ran for roughly two hours. They were then removed and placed in the -20C fridge.

Along with this we also communicated with Chad regarding the multiple range of buffer solutions needed for the upcoming pH experiment. Above I mentioned the experiment, where we will be exposing d. ficus to a range of pHs to confirm it's ability to survive in such a high alkaline environment.  

  Wednesday 10.23.24

  Gel was performed on 4 PCR reactions started the latter day, to reiterate these were to test WT D. ficus against both sets of primers. AMP and CMR. In this gel we compared them against the plasmid, pRAD1, and found that the WT may have homologous binding attaching to other parts of chromosomal or plasmid DNA found natively in d. ficus. Moving forward we are going to perform a plasmid extraction on the transformed d. ficus to confirm the pRAD1 plasmid is there. 

The idea of fresh primers being test against and made has come up, but we want to see what happens after the plasmid extraction and post PCR.

Thursday 10.24.24

        Analyzed gels and discussed potential next move, where we will be doing a plasmid extraction on         WT ficus, our potential transformed ficus and the prad1 extraction, using both CMR and AMP                primers. 

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