This step entailed the extraction of plasmid (pBKS II) fromÂ E. coli. First, 1.5 ml of previously prepared bacterial culture was centrifuged at maximum speed for 1 min and the supernatant poured off to generate a dry pellet containing bacterial cells. This was followed by a re-suspension of the pellet in GTE buffer before swirling to generate a homogeneous mixture. A lysis buffer (1% SDS and 0.2 M NaoH) was added to the mixture to lyse the cells and release their contents. The high PH created was lowered using a neutralization buffer (3 M Potassium acetate). The mixture was then centrifuged at maximum speed for 10 minutes. Subsequently, 400 Âµl of the supernatant was transferred into another tube.
To precipitate the DNA, 1000ÂµL of 100% ethanol was added to the contents of the tube. This was followed by centrifugation at top speed for 10 min to separate genomic DNA (the pellet) from DNA-binding proteins. The pellet was washed in 1ml and again in 500Âµl of 70% ethanol. This was followed by centrifugation at top speed first for 2 min and then for15 min. The pellet generated was dried using paper towels before being re-suspended in Tris-HCl buffer containing RNase A. The mixture was then incubated at 37Â°C for 10 min to denature RNA. The pellet was stored on ice for the next step.
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