Team:Macquarie Australia/WetLab/Protocols/PlasmidPreps

Plasmid Prep

• Centrifuge @13,200 rpm for 10 min of 1.8mL of overnight cultures in 2mL Eppendorf’s
• Discard supernatant and add 1.9 mL of culture and centrifuge again @13,200 rpm for 10 min
• Re-suspend pelleted cells in P1 Buffer (250µl)
• Add 250µL of P2 buffer & invert 4-6 times (turns homogenous blue)
• Add 350µL of N3 & mix by inverting (turns colourless)
• Centrifuge @ 10min 13,000 rpm to obtain a pellet
• Transfer supernatant in QIA Prep Spin Column by pipetting
• Centrifuge 30-60 sec - discard flow through
• Wash QIA Prep Spin Column with 0.5mL of PB
• Centrifuge for 30-60 seconds - discard flow through
• Wash spin column by adding 0.75 mL PE buffer
• Centrifuge for 30-60 seconds - discard flow through and centrifuge for a further 1min
• Place QIAPrep Column in clean Eppendorf
• Elute DNA add 50µl of water, stand for 1min, centrifuge for 1min
• QIA prep - Spin miniprep buffer

Figure 1. Plasmid prep for QIAGEN QIAprep Spin Miniprep Kit.