Griffitts:Miniprep: Difference between revisions
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==Procedure== | ==Procedure== | ||
* Grow DH5α with appropriate plasmid in liquid LB | * Grow DH5α with appropriate plasmid in 4 mL liquid LB+[[Griffitts:Stock_solutions#Antibiotics|antibiotic]] for 12–18 hours | ||
* Label your spin columns and microcentrifuge tubes | * Label your spin columns and microcentrifuge tubes | ||
* Centrifuge 1. | * Centrifuge 1.6 mL of each culture in a microcentrifuge tube at full speed for 30 seconds | ||
* Dump and tap out supernatant | * Dump and tap out supernatant | ||
* Resuspend pellet in 250 μL P1 | * Resuspend pellet in 250 μL P1 | ||
** The P1 is kept in the fridge | ** The P1 is kept in the fridge | ||
| Line 28: | Line 25: | ||
* Centrifuge for 30 seconds at full speed | * Centrifuge for 30 seconds at full speed | ||
* Remove flowthrough | * Remove flowthrough | ||
* Add | * Add 700 μL of wash buffer PE to column | ||
* Centrifuge for 30 seconds at full speed | * Centrifuge for 30 seconds at full speed | ||
* Remove flowthrough | * Remove flowthrough | ||
* Centrifuge for another 60 seconds at full speed | * Centrifuge for another 60 seconds at full speed | ||
* Move column to a labeled 1.5 mL microcentrifuge tube | * Move column to a labeled 1.5 mL microcentrifuge tube | ||
* Add 50 | * Add 50–100 μL TE directly to disk | ||
** Don't touch the membrane with your tip | ** Don't touch the membrane with your tip | ||
* Let stand 1 | * Let stand 1–2 minutes | ||
* Centrifuge for 60 seconds at full speed to elute DNA | * Centrifuge for 60 seconds at full speed to elute DNA | ||
''Adapted from Qiagen handbook'' | ''Adapted from Qiagen handbook'' | ||
Latest revision as of 09:56, 16 May 2011
Standard Qiagen Miniprep
Materials
- Qiaprep Spin Kit (27106)
- TE buffer
- 1.5 mL microcentrifuge tubes (1 per culture)
Procedure
- Grow DH5α with appropriate plasmid in 4 mL liquid LB+antibiotic for 12–18 hours
- Label your spin columns and microcentrifuge tubes
- Centrifuge 1.6 mL of each culture in a microcentrifuge tube at full speed for 30 seconds
- Dump and tap out supernatant
- Resuspend pellet in 250 μL P1
- The P1 is kept in the fridge
- Make sure to use P1 that has RNAse added
- Add 250 μL P2
- Immediately invert 3 times
- Let stand for 2 minutes
- Add 350 μL N3
- Immediately invert 8 times
- Centrifuge for 6 minutes at full speed
- Carefully move the supernatant (~820 μL) to a spin column
- Centrifuge for 30 seconds at full speed
- Remove flowthrough
- Add 700 μL of wash buffer PE to column
- Centrifuge for 30 seconds at full speed
- Remove flowthrough
- Centrifuge for another 60 seconds at full speed
- Move column to a labeled 1.5 mL microcentrifuge tube
- Add 50–100 μL TE directly to disk
- Don't touch the membrane with your tip
- Let stand 1–2 minutes
- Centrifuge for 60 seconds at full speed to elute DNA
Adapted from Qiagen handbook
