IIT Madras

And then the E.coli said, "Let there be light"

Preperation of competent cells - DH5

• Grow in 2 test tubes (5 ml LB broth in each with required antibiotic) transformed cells for about 12 – 16 hours (Max 16 hours).
• Pellet down the cells in 1 eppendorf (each) 1000 rpm, 4 C, for 2 minutes.
• Add 250 ul of Buffer P1 (ice cold). Resuspend the pellet. Wait for 2-5 minutes (Room temperature).
• Add 250 ul of Buffer P2. Invert mix. Waif for 2-5 minutes (Room temperature)
• Add 350 ul of (ice cold) neutralization buffer (HiPura); Invert mix. Incubate on ice for 5 minutes.
• Centrifuge for 10 min at 12000 rpm 4 C.
• Collect supernatant and apply to QIAprep spin column. Centrifuge for 30 – 60 s. Discard flow through.
• Add 750 ul of Wash buffer. Spin and discard the flow through.
• Centrifuge an additional 1 min to remove any residual wash buffer.
• Place the column in a 1.5 ml microcentrifuge tube. To elute DNA, add 50 ul of Buffer EB or water at the center of each column, let it stand for 2 minutes. Incubate at 65 C for 2 mins in case of low copy number plasmids.

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