Week of 10/7/24

 Our R2 primer worked for ligation confirmation, however A1/A2 primers did not work because the annealing temperature may have been too high which caused a half ligated PgRNA and Kana fragment which Xbai could not ligate. We decided to do overnight ligation of PgRna and Xbai. We were also able to confirm that each enzyme (Xbai and BAM) worked separately on the fragment, however in conjunction they did not. Considering our R2 primer worked this would mean that there may be a digestion issue of Xbai. We also performed plasmid extraction on e.coli pdCas9 and transformed sonora with such. We made competent cells of pdCas9 as well. 

Comments

Post a Comment

Popular posts from this blog

Week of 11/22/24 pH Experiment on Sonora's Biofilm Breakdown

Image
 After incubating the pH range of 6,7, and 8 the cells were gram stained after 24 hrs along with a plain TGY broth containing Sonora for a comparison and all of the flasks were homogenized strangely enough. The morphology was still irregular as if the cells were still dying. The gram stains albeit were not completely viable, as there was a variable gram negative to gram positive ratio. The OD values were taken as well, and the results were also varied as pictured below.
Read more

Week of 11/18/24 Breakdown of sonora's Biofilm via pH Experiment

 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. 
Read more

Week of 11/4/24

 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. 
Read more