Kafatos:Minipreps: Difference between revisions
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===Day 2=== | ===Day 2=== | ||
- spin 1.5 ml 2’ @ 8,000 rpm in table top centrifuge<br> | - spin 1.5 ml 2’ @ 8,000 rpm in table top centrifuge<br> | ||
- resuspend pellet in 100 | - resuspend pellet in 100 μl [[Kafatos:P1|P1 Buffer]]. Let sit 5’ @ RT<br> | ||
- add 200 μl P2 Buffer, mix and let sit no more than 5’ @ RT<br> | - add 200 μl [[Kafatos:P2|P2 Buffer]], mix and let sit no more than 5’ @ RT<br> | ||
- add 150 μl ice cold P3 Buffer, mix well and place 5 – 10’ on ice<br> | - add 150 μl ice cold [[Kafatos:P3|P3 Buffer]], mix well and place 5 – 10’ on ice<br> | ||
- spin 10’ @ max rpm (4˚C if possible)<br> | - spin 10’ @ max rpm (4˚C if possible)<br> | ||
- transfer supernatant to new tube and add 2 volumes of abs EtOH. Let sit for 10 – 20’ on ice<br> | - transfer supernatant to new tube and add 2 volumes of abs EtOH. Let sit for 10 – 20’ on ice<br> | ||
Revision as of 04:17, 30 May 2006
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Day 1
- inoculate 5 ml sterile LB medium (+ antibiotic ?) with single bacterial colony
- grow ON @ 37˚C while shaking vigorously
Day 2
- spin 1.5 ml 2’ @ 8,000 rpm in table top centrifuge
- resuspend pellet in 100 μl P1 Buffer. Let sit 5’ @ RT
- add 200 μl P2 Buffer, mix and let sit no more than 5’ @ RT
- add 150 μl ice cold P3 Buffer, mix well and place 5 – 10’ on ice
- spin 10’ @ max rpm (4˚C if possible)
- transfer supernatant to new tube and add 2 volumes of abs EtOH. Let sit for 10 – 20’ on ice
- spin 10’ @ max. speed @ 4˚C
- discard supernatant and wash pellet with 1ml of 70% EtOH
- spin 5’ @ max speed
- suck up or decant supernatant, speedvac or dry pellet @ 37˚C (do not overdo it, though, or it will not properly dissolve again)
- resuspend pellet in 30 μl TE Buffer or ddH20 and check on gel
note: plasmids obtained with this protocol are not clean enough for sequencing
