Great Kit From Qiagen for Bacterial Plasmid DNA Extraction

We use this kit daily in my lab. Simple and fast, it yields high-quality DNA for any downstream applications. After the transformation of TOP10 E. coli competent cells, I picked 3 colonies for confirmation by Sanger sequencing. Colonies were inoculated in 3 mL of LB medium and incubated at 37 °C, 220 rpm for 6 h. After that, I proceeded to plasmid extraction. My plasmid is 14 kb long, a Dengue virus infectious clone. Using this kit, I can extract DNA in less than 30 minutes. I usually follow the supplier's protocol. The bacteria cells were centrifuged and resuspended in 250 uL of Buffer 1, and mixed with 250 uL of Buffer 2. I usually incubate my samples at room temperature for 3 minutes in this step, to allow cell lysis. Next, I added 350 uL of Buffer 3 and centrifuged the samples for 10 min at 13000 rpm. Finally, I pass my lysate through the column using a vacuum manifold and wash the column with Buffer PE (which contains ethanol). I eluted my DNA in 35 uL of deionized water. DNA concentration was 185 ng/uL, which I considered a good amount considering a 3 mL culture that grew for 6 hours. 260/280 ratio was also good. Overall, it is a good and reliable kit.