Week of 11/12/24 Plasmid Extraction and Lambda Digestion

 Intro:

Due to our prior attempts with plasmid extraction of e.coli PdCas9 it was decided that we use fresh e.coli PdCas9 that had been inoculated within 24hrs in order to achieve optimal OD values for the use of transformation of sonora.We also found that we had contamination with our PdCas9 transformation of e.coli. 

Methods:

Going forward we inoculated 20ml of LB broth (CMR 25ug/ml) with retransformed PdCas9 e.coli to use for the plasmid extraction as opposed to inoculating the culture epindorph tube from plates. As for the continuation of establishing succesful ligation we also had planned out the schedule for Xbai digestion. In order to narrow down the problem with the ligation of Xbai on PgK it was decided that we will digest lambda with Xbai and see on a gel if it is the primers or the digestion itself that is the issue with ligation. To do this, we had used the big electrophorisis gel because we needed to see a clearer distinction between the bands of the 1KB ladder extended, lambda DNA, and Xbai digested Lambda. I also had brainstormed for my Spring conference poster project, in which fine tuning had been made to the methods according to the overarching idea of sonora's biofilm effecting antibiotic resistance of Tetracycline. 

Results:








Discussion:

We found out that there had been some user errors with the plasmid extractions lately, as DI water had been used in place of the culture broth which had resulted in the cells lysing due to the hypotonic solution of DI water. Upon reattempting plasmid extraction with the above, our OD values were still abysmal. We are not sure why, this could be due to the fact we did 20ul of elution instead of the 30ul because we wanted to get more DNA, it could be that the plasmid extraction needs time to sit before taking the nanodrop value, or that the e.coli is holding onto a low copy of the plasmid.

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