S-STEM Scholar Madi Cook's Blog

 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. 

 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. 

 We did PCR on the ligated Kanamycin fragment in order to replicate more of our sample to able to load it onto a gel for confirmation and future uses. We also transformed e.coli with the PCR product of the Kana fragment so that way we can perform plasmid extraction and transform sonora with the kana fragment as well. We also redid double digestion once more just to ensure that it is the Xbai that is the problem in conjunction with the BAM fragment. We did this via a 3:1 molar ratio of insert to vector as the New England protocol for double digestion calls for. 

In order to assemble the PgK (PgRNA and Kanamycin) fragment onto the guide plasmid, we still needed to have successful double digestion of PgRNA with enzymes Xbai and BAM. BAM has worked without failure, but we are inhibited in our progress due to the Xbai fragment not ligating despite the primers being coded specifically for this fragment. We are not sure why this may be, so we are narrowing down on what exactly is the problem whether or not the primers are bad, if the enzyme itself is bad, or if the double digestion is not suitable for Xbai to ligate. Going forward, we will only single digest Xbai and find a basis for which the issue is. We have also brainstormed a name for our plasmid, PVELM (Plasmid and the first initial of the prime researchers within our group that have been working on this since summer, Vanshika, Emme, Lani, Madi). 

 We passaged PWT-Cas9 onto plates with TGY and varying amounts of Ampicillin concentrations. The concentration was 0.4 ug/ml on two plates, 0.6ug/ml on two plates, and 0.8ug/ml on two plates. This was the method in which we decided on verifying for successful transformation of D. sonora. However, upon doing this passage we discovered that using PWT-Cas9 would not be viable to use in conjunction with sonora due to its innate insanely high tetracycline resistance. We are deciding on moving forward using PdCas9 as we are able to use Chloremphenicol instead.