week 9/24-27

  • This week was focused more on the transformation of Kanamycin and PGRNA.
    • kanamycin cleanup 
      • NEB monarch cleanup kit 
        • added 260 µL of binding buffer to 52 µL of sample (5:1 ratio) 
        • 15 ul of ellusion

        • The nanodrop read 74.1 ng/µL with an A260/A280 concentration of 1.84 and an A260/A230 concentration of 1.13.

        • Run gel on kana after cleanup.
          • 900 base pairs  
      • double digest after cleanup 
        • After digesting kanamycin and PGRNA, set up ligation. 
        • If ligation looks good, need to immediately place in E. coli.
    • gram stain PGRNA E. coli
    • transformation of pdcas-9 
      • 2 tubes, one with competent cells and a control tube with no competent cells 
        • If transformation was successful, cells should be resistant to ampicillin. 
    • Sonora D. is resistant to tetracycline.
    • A plasmid is a small piece of DNA with essential genes like antibiotic resistance. 
    • PCR
      • polymer chain reaction 
          • give templates for primers to replicate  

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