S-STEM Scholar Madi Cook's Blog

 In order to move forward with determining how to breakdown Sonora's persistent biofilm, a trial run of using varying pH values (pH 6, 7, 8) with 20mMolar MOPS buffers in TGY broth were made. Upon inoculating Sonora and incubating in the -30 degree shaking incubator for 72hrs, all of the flasks of varying pH had homogenized Sonora. The gram stains had revealed that it was in fact Sonora, yet the morphology was irregular as if the cells were dying. Going forward the trial will be adjusted to a incubation time of 24hrs rather than 72hrs in order to determine if it is the buffers that are effecting the cells or simply the incubation time. 

 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 ex...

 A gram stain on pdCas9 e.coli was performed in which it came out to be contaminated we think this may be due to the parent plate having been contaminated. So we are retransforming e.coli with pdCas9 and then confirming that contamination did not take place. In the event that we had e.coli with pdCas9 confirmed, we did a freezeback of it so as to have a source. 

 Moved forward with single digestion of Kana fragment using Xbai. D. sonora plain was also passaged onto TGY and R2A. Our pdCas9 of Sonora was contaminated, so we have to redo transformation and possibly plasmid extraction. For single digestion of PgRNA it was digested and ligated with Xbai first then BAM digestion and ligation.

 We redid PCR attempt due to a discrepancy of verification. We also did plasmid extraction in order to get more sample of Pdcas9. We are deciding if a new verification process to narrow down the issue with Xbai ligation using lambda would be viable. More chloremphenicol plates for passaging e.coli pdcas9 were made. 

 When we ran the gel on Pgk and PgRNA we found that both were about the size of 200bp, which means that they were not successful. So we did religate PgK. We did another gel just to confirm these results due to the fact the 1KB ladder was not viable. We also increased our insert to vector molar ratio to 5:1 to have a better chance of the insert coming together with the vector.