When editing bacteria with CRISPR, less is more NatureComms
; pDC786, pDC841, pDC876, pDC869, pDC829, pDC860, pDC889, pDC862, and pDC886) and selected for on appropriate antibiotics. Individual colonies were inoculated into LB with appropriate antibiotics and incubated at 37 °C shaking at 220 rpm overnight. Cultures were then back-diluted into 15 mL of fresh LB with antibiotics and grown to an ABSof 0.6–0.8. 5 mL of this culture was mixed with 1 mL of STOP mix, 95% of 100% ethanol and 5% hot phenol .
The samples were then spun down at 13,000 rpm at 12 °C for 15 min. The upper phase was then transferred into a sterile 2 mL Eppendorf tube where 1.4 mL of 30:1 ethanol: sodium acetate pH 6.5 was added. The RNA was precipitated at −20 °C for 2 h. The samples were then spun down at 13,000 rpm at 4 °C for 30 minutes. The supernatant was removed and 500 µL of 70% ethanol was added to the pellet. The samples were spun down at 13,000 rpm at 4 °C for 10 min.
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