GRS Plasmid Purification Kit - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki

GRS Plasmid Purification Kit

Manual PDF

  1. Transfer 1,5 ml of a culture of bacterial cells harbouring the plasmid of interest to a 1,5-ml microcentrifuge tube and harvest the cells by centrifugation at 14- 16.000g for 1 minute. Discard the supernatant. If desired, more than 1,5 ml of cultured cells (up to 6 ml) can be used by repeating the harvesting step.

  2. Add 200 μL of Buffer P1 (check if RNAse A was added !) to the bacterial pellet, and resuspend the cells by vortexing or pipetting up and down.

  3. Add 200 μL of Buffer P2 (check for precipitates) and mix immediately by gently inverting the tube 10 times. DO NOT VORTEX! (vortexing might lead to shearing of genomic DNA). Incubate at room temperature until lysis is complete.

Note that after adding buffer P2, precipitates will completely dissolve and the suspension will become blue. Continue mixing in case the suspension contains brownish cell clumps or colourless regions (see pictures on page 2)

  1. Neutralize the solution by adding 300 μL of Buffer P3. Mix immediately by gently inverting the tube 10 times. DO NOT VORTEX! Note that after adding buffer P3, the suspension becomes colourless. Continue mixing in case the suspension contains blue regions (see pictures on page 2). Centrifuge at 14-16.000g for 3 minutes. DNA Binding

  2. Place a Plasmid DNA Mini Spin Column into a 2,0-ml collection tube. Add the supernatant from step 5 to the column. Centrifuge at 14-16.000g for 30 seconds.

  3. Discard the flow-through, and place the spin column back in the collection tube. Add 400 μl of Wash Buffer 1 and centrifuge at 14-16.000g for 30 seconds.

  4. Discard the flow-through, and place the spin column back in the collection tube. Add 600μl of Wash Buffer 2* (check if ethanol is added)

  5. Centrifuge at 14-16.000g for 30 seconds and discard the flow-through

  6. Place the spin column back in the collection tube and centrifuge at 14-16.000g for another 3 minutes to dry the matrix of the column.

  7. Transfer the spin column to a new 1,5-ml microcentrifuge tube and pipet 50μl Elution Buffer directly to the centre of the spin column without touching the membrane. Incubate at room temperature for 2 minutes. Notes: Yield could be increased using pre-warmed Elution Buffer (60oC-70oC). Instead of Elution Buffer, DNA can also be eluted with TE or water; pH should be 8.0-8.5.

  8. Centrifuge for 2 minutes at 14-16.000g to elute purified DNA. Discard the spin column and use DNA immediately or store at -20oC.

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