Kafatos:Minipreps

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 7: Line 7:
===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 l P1 Buffer. Let sit 5’ @ RT<br>
+
- 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 07:17, 30 May 2006

Click here to visit our NEW WEBSITE
The content below is most likely out of date. We also have a new lean and mean openwetware area.


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

Personal tools