November 4th-November 8th

 Week 7 (Nov 4th-Nov 8th)

This week, we concluded that our gram stain of pdCas9 and e. coli showed contamination which we were worried was staff. We decided to gram stain our parent plate and another member of the lab’s plate and decided if that second plate shows no contamination, we’ll inoculate onto two chloramphenicol plates.


Plate A (parent plate) looked contaminated and Plate B (other lab plate) looked okay. The next day, the 5th, we decided to gram stain the two e coli plates we inoculated and found that whatever it was, it was not the e coli we were looking for. Generally, we have no idea what happened but it wasn't e coli with pdcas 9 and instead something completely else than we initially thought it was, although the bacteria is still unknown.


We decided to take an old plate of pdcas 9 e coli and run a gel on that using 2 ul of dna, 8 ul of water, and 2 ul of dye at a concentration of 42.4 ng/ul at 50 amps. 


On the sixth, we performed a transformation of pdcas9 and e coli with sonora and made plans to look for growth the next day along with discussed making a freezeback of the pdcas9 with E. coli..


Transformation Protocol

  1. That competent cells in cryovial on ice.

  2. Aliquot 100ul of competent cells into a micro centrifuge tube.

  3. Add 1ug or <10ul of plasmid solution into the micro centrifuge tube.

  4. Place on ice for 15 minutes.

  5. Place in incubator at 30°C-32°C for 45 minutes, agitating every 15 minutes by inverting tubes.

  6. After transferring content into 15ml centrifuge tubes with 4 ml of TGY broth.

  7. Incubate into a shaking incubator at 30°C for 16 hours.

  8. After, plate onto TGY agar with antibiotics overnight for four days.

  9. Observe growth on plates.


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