week 10/30 -11/4

LB Broth Preparation and Transformation:

  • LB broth was made for the growth and transformation of E. coli.

Overnight Ligation:

  • Ligation of kanamycin and pgRNA was planned for overnight because of past unsuccessful ligations.

Quick Ligation Protocol:

  • Quick Ligation Buffer: 10 µl used to maintain pH, preventing the ligase from degrading pgRNA.

  • Kanamycin Calculation: Based on the online calculator, 177.8 ng of kanamycin was needed, based on a 5:1 ratio.

  • pgRNA: 2 µl of 50 ng/µl concentration was used for a total of 100 ng.

  • Kanamycin: 3.5 µl of 50 ng/µl concentration was used for a total of 177.8 ng.

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