Turkey/ Protocols
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| - | Growing colonies in broth: | + | '''Growing colonies in broth:''' |
1. Prepare 5mL LB + Amp broth in a sterile Falcon tube. | 1. Prepare 5mL LB + Amp broth in a sterile Falcon tube. | ||
| Line 5: | Line 5: | ||
3. Incubate at 37C incubator for 14-16 hours. | 3. Incubate at 37C incubator for 14-16 hours. | ||
| - | Miniprep Plasmid Isolation (with Qiagen kit): | + | '''Miniprep Plasmid Isolation (with Qiagen kit):''' |
1. Centrifuge the falcons at 4000rpm for 4-10 minutes. | 1. Centrifuge the falcons at 4000rpm for 4-10 minutes. | ||
| - | 2.Resuspend pelleted bacterial cells in 250ML Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf. | + | 2. Resuspend pelleted bacterial cells in 250ML Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf. |
| - | 3.Add 250ML Buffer P2 and invert the tube for 6 times to mix (do not vortex). | + | 3. Add 250ML(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added). |
| + | 4. Add 350ML Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy. | ||
| + | 5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed. | ||
| + | 6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through. | ||
| + | 7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min. | ||
| + | 8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer. | ||
| + | 9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32ML (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the C [[center]] of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min. | ||
| + | |||
| + | Store the minipreps at -20. The concentration obtained can be measured by Nanodrop. | ||
Revision as of 21:35, 13 August 2007
Growing colonies in broth:
1. Prepare 5mL LB + Amp broth in a sterile Falcon tube. 2. Pick up a colony from the plate by a micropipette tip or sterile toothpick and put it in the falcon. 3. Incubate at 37C incubator for 14-16 hours.
Miniprep Plasmid Isolation (with Qiagen kit):
1. Centrifuge the falcons at 4000rpm for 4-10 minutes. 2. Resuspend pelleted bacterial cells in 250ML Buffer P1(kept in +4C) and transfer to a 1.5mL eppendorf. 3. Add 250ML(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added). 4. Add 350ML Buffer N3 and invert the tube immediately for ~6 times. Solution should become wtite and cloudy. 5. Centrifuge for 10 minutes at 13000 rpm. A compact white pellet will be formed. 6. Pour the supernatant to Qiagen spin column and centrifuge the column for 1 min. Discard the flow-through. 7. Wash the spin column by adding 0.75mL Buffer PE and centrifuge for 1 min. 8. Discard the flow-through and centrifuge for an additional 6.5-7min to remove residual ethanol in the wash buffer. 9. Place the Qiagen prep column in a clean 1.5 mL eppendorf. Add 32ML (can be modified according to the concentration aimed to be obtained) Buffer EB or water to the C center of each Qiagen prep spin column and let stand for 5-10 minutes. Centrifuge for 1 min.
Store the minipreps at -20. The concentration obtained can be measured by Nanodrop.
