for
DNA Extraction - Plasmid - MoBio
Please
note: The
checklist assumes that you are familiar
with
basic techniques and just need to know what the steps are!Ý
Refer to the complete protocols if you need more information.
Revised May
2002
___ 1. ÝCentrifuge 2 mL of culture for 1 minute at maximum g.Ý (Keep it at this setting.)
___ 2. ÝDiscard supernatant.
___ 3. ÝCentrifuge for 30 seconds.
___ 4. ÝDiscard any remaining supernatant.
___ 5. ÝAdd 50 mL of Solution 1, then resuspend pellet by vortexing 4 or 5 times for 2 seconds at a time until completely resuspended.
___ 6. ÝAdd 100 mL of Solution 2, then invert once to mix Ý(Inverting more than once may result in contamination by chromosomal DNA.)
___ 7. ÝAdd 325 mL of Solution 3, then invert once to mix.
___ 8. ÝCentrifuge for 1 minute.
___ 9. ÝTransfer supernatant into a clean spin filter (do not contaminate with the pellet.)
___ 10. ÝCentrifuge for 30 seconds.
___ 11. ÝRemove the spin filter, discard the supernatant (flow-through), then replace the spin filter.
___ 12. ÝAdd 300 mL of Solution 4 into the spin filter.
___ 13. ÝCentrifuge for 30 seconds.
___ 14. ÝDiscard supernatant (avoid contact with the spin filter)
___ 15.Ý Centrifuge for 30 seconds.
___ 16. ÝPlace the spin filter into a new 2.0 mL tube.
___ 17. ÝAdd 50 mL of autoclaved Nanopure water (or Solution 5) into the spin filter.Ý
___ 18. ÝCentrifuge for 30 seconds.
___ 19. ÝDiscard the spin filter.Ý The supernatant flow-through contains the plasmid!