Plasmid DNA extraction from e.coli for digestion mapping clony screening

Plasmid DNA extraction from e.coli for digestion mapping clony screening:

This protocol utilize the Qiagen buffer P1, P2 and P3 from Qiagen
plasmid purification kit. RNase must be added before use. It is cost
efficient since it doesn't use expensive plasmid purification columns.
This protocol is only intended for screening positive clony. Once
positive clonies are determined, purification from bacterial overnight
culture can be done using plasmid purification kit for downstream
applications.

1. Culture e.coli overnight @ 37c with vigorous shaking.
2. Take 1.5 ml of o/n culture in fresh eppendorf tube.
3. Spin down at 2500 rpm x 10min.
4. Thoroughly remove all medium using a very thin tip pasteur glass
pipette connected to vacuum. Resuspend bacterial pellet in 300 ul
buffer P1, vortex to resuspend.
5. Add 300 ul of buffer P2, mix GENTLY by invert tubes several times
and incubate at room temperature for 5 min.
6. Add 300 ul of buffer P3, mix immediately and GENTLY, incubate on
ice for 10 min.
7. 13,000 rpm x 10min at room temperature.
8. Carefully transfer supernatant in a new eppendorf tube (~ 800ul).
9. Add 560 ul of 100% isopropanol and mix gently.
10. Incubate at room temperature for 30 min.
11. 13,000 rpm x 30 min @ 4c to pellet the DNA precipitation.
12. Wash DNA pellet with 70% ethanol (don't disturbe pellet) once time.
13. 13,000 rpm x 5 min to spin down DNA pellet.
14. Remove ethanol wash solution, air dry for 5-10min at room
temperature. Be sure not to overdry the DNA pellet.
15. Add 20 ul miniQ water to dissolve DNA pellet.
16. Set up restriction digestion cocktail for restriction enzyme(s)
digestion (1 hour).
17. Run analytical agarose gel (30min).

Archived page

Last update 19-Apr-2007, Rating Good of 1 votes.