Difference between revisions of "Shreffler:Notebook/retinoic acid trans signaling/2009/05/20"

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( Note: This process is used for rapid purification of transfection grade plasmid DNA)
 
( Note: This process is used for rapid purification of transfection grade plasmid DNA)
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temperature for 5 min
 
temperature for 5 min
  
4.  
+
- 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
 +
 
 +
4. Add 10 ml of chilled Buffer P3, mix throughly by vigorously inverting 4-6 times
 +
 
 +
 
 +
B) Bacterial lysate clearing
 +
 
 +
5. 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!
 +
 
 +
6. 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
  
  

Revision as of 11:29, 1 June 2009

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Plasmid Purification:

( Note: This process is used for rapid purification of transfection grade plasmid DNA)



Kit: Qiagen: HiSpeed Plasmid Purification Maxi Kit

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

- 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

4. Add 10 ml of chilled Buffer P3, mix throughly by vigorously inverting 4-6 times


B) Bacterial lysate clearing

5. 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!

6. 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