Shreffler:Plasmid Purification

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(New page: ==Overview== ==Materials== * Kit: Qiagen: HiSpeed Plasmid Purification Maxi Kit ==Procedure== A) Bacterial culture, harvest, and lysis # Pellet 150 ml overnight LB culture at 6000 x g ...)
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Contents

Overview

Materials

  • Kit: Qiagen: HiSpeed Plasmid Purification Maxi Kit

Procedure

A) Bacterial culture, harvest, and lysis

  1. Pellet 150 ml overnight LB culture at 6000 x g for 15 min at 4 degree C
  2. Homogeneously resuspend the bacterial pellet in 10 ml Buffer P1
  3. Add 10 ml Buffer P2, mis throughly by vigoorously inverting 4-6 times, and incubate at room temperature for 5 min

Note:

  • During the incubation prepare the QIAfilter Cartrigde
  • Screw the cap onto the outlet nozzle of the QIAfilter Maxi Cartridge
  • Place the QIAfilter Cartridge into a convenient tube or QIArack
  1. Add 10 ml of chilled Buffer P3, mix throughly by vigorously inverting 4-6 times

B) Bacterial lysate clearing

  1. Pour the lysate into the barrel of the QIAfilter Catridge, and incubate at room temperature (15-20 degrees C) for 10 min, do not insert the plunger!
  2. Equilibrate a HiSpeed Maxi Tip by applying 10 ml Buffer QBT and allow the column to empty by gravity flow

7. Remove the cap from the QIAfilter Cartridge outlet nozzle, gently insert the plunger into the QIAfilter Maxi Cartridge and filter the cell lysate into the previously equilibrated HiSpeed Tip


C) Bind, wash,, and elute plasmid DNA on HiSpeed Tip

8. Allow the cleared lysate to enter the resin by gravity flow

9. Wash the QIAGEN-tip with 60 ml of Buffer QC

10. Elute DNA with 15 ml of Buffer QF


D) Precipitate, wash, and redissolve plasmid DNA

11. Precipitate DNA by adding 10.5 ml of room-temperature isopropanol to the eluted DNA, mix and incubate for 5 min

12. During the incubation remove the plunger from a 30 ml syringe and attach the QIAprecipitator Maxi module onto the outlet nozzle

13. Place the QIAprecipitator over a waste bottle, transfer the eluate/isopropanol mixture into the 30 ml syringe, and insert the plunger; filter the eluate/isopropanol mixture through the QIAprecipitator using constant pressure

14. Remove the QIAprecipitator from the 30 ml syringe and pull out the plunger. Re-attach the QIAprecipitator and add 2 ml 70% ethanol to the syringe. Wash the DNA by inserting the plunger and pressing the ethanol through the QIAprecipitator using constant pressure.

15. Remove the QIAprecipitator from the 30 ml syringe and pull out the plunger. Attach the QIAprecipitator to the 30 ml syringe again, insert the plunger, and dry the membrane by pressing air through the QIAprecipitator quickly and forcefully. Repeat this step.

16. Dry the outlet nozzle of the QIAprecipitator with absorbent paper to prevent ethanol carryover

17. Remove the plunger from a new 5 ml syringe and attach the QIAprecipitator onto the outlet nozzle. Hold the outlet of the QIAprecipitator over a 1.5 ml collection tube. Add 1 ml of Buffer TE to the 5ml syringe. Insert the plunger and elute the DNA into the collection tube using constant pressure.

18. Remove the QIAprecipitator from the 5 ml syringe, pull out the plunger and reattach the QIAprecipitator to the 5 ml syringe.

19. Transfer the eluate from 17 to the 5 ml syringe and elute for a second time into the same 1.5 ml tube


Discussion

discuss this protocol

References

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Qiagen HiSpeed Maxi Kit Prep Protocol:

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