Griffitts:Low-copy plasmid prep: Difference between revisions
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* 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 70 μL TE (preheated to 65°C) directly to disk | * Add 70 μL [[Griffitts:Common buffers#TE buffer]] (preheated to 65°C) directly to disk | ||
** Don't touch the membrane with your tip | ** Don't touch the membrane with your tip | ||
**Note that TE must be added to the column hot for efficient elution of large (20 kb) plasmids | **Note that [[Griffitts:Common buffers#TE buffer]] must be added to the column hot for efficient elution of large (20 kb) plasmids | ||
* Let stand 1-2 minutes | * 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 |
Revision as of 11:39, 13 February 2008
Miniprep procedure for large, low-copy plasmids
Materials
Procedure
Lysis
- Set up an overnight culture in 40 mL LB broth
- Centrifuge for 8 min at 8000 rpm
- Resuspend in 2.5 mL of resuspension buffer
- Add 2.5 mL of lysis solution
DNA Isolation
- Add 2.5 mL of neutralization solution
- Centrifuge at 13,000 rpm for 8 min
- Transfer 6.5 mL of supernatant to 15-mL Falcon tube
- Add 50 μL of RNAse A
- Let sit at RT for 5 min
- Add 6.5 mL of isopropanol
- Incubate at -20°C for 30 min
- Centrifuge in clinical (yellow) centrifuge for 10 min at max speed
- Pour off supernatant
- Resuspend pellet in 1 mL 70% ethanol
- Centrifuge for 5 min in clinical centrifuge (yellow) at max speed
- Discard supernatant
Qiagen Miniprep
- Resuspend DNA pellet in 250 μL of Buffer P1
- The P1 is kept in the fridge
- Make sure to use P1 that has RNAse added
- Add 250 μL of Buffer P2
- Immediately invert 3 times
- Let stand for 2 minutes
- Add 350 μL of Buffer N3
- Immediately invert 8 times
- Keep on ice for 5 min
- Centrifuge at 13,000 rpm for 6 min in microfuge
- 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 70 μL Griffitts:Common buffers#TE buffer (preheated to 65°C) directly to disk
- Don't touch the membrane with your tip
- Note that Griffitts:Common buffers#TE buffer must be added to the column hot for efficient elution of large (20 kb) plasmids
- Let stand 1-2 minutes
- Centrifuge for 60 seconds at full speed to elute DNA