Shreffler:Notebook/retinoic acid trans signaling/2009/05/20: Difference between revisions
No edit summary |
No edit summary |
||
| Line 16: | Line 16: | ||
Kit: Qiagen: HiSpeed Plasmid Purification Maxi Kit | Kit: Qiagen: HiSpeed Plasmid Purification Maxi Kit | ||
Maxi Kit Prep Protocol: | |||
A) Bacterial culture, harvest, and lysis | A) Bacterial culture, harvest, and lysis | ||
| Line 41: | Line 43: | ||
6. Equilibrate a HiSpeed Maxi Tip by applying 10 ml Buffer QBT and allow the column to empty by gravity flow | 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 | 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 | |||
Revision as of 14:48, 1 June 2009
 Retinoic Acid Trans Signaling
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Plasmid Purification: ( Note: This process is used for rapid purification of transfection grade plasmid DNA)
Maxi Kit Prep Protocol: 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
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 nozzle, gently insert the plunger into the QIAfilter Maxi Cartridge and filter the cell lysate into the previously equilibrated 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
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
| |
