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. |
| - | 2. Pick up a colony from the plate by a micropipette tip or sterile toothpick and put it in the falcon. | + | *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. | + | *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(microliter) Buffer P2 and invert the tube for ~6 times to mix (do not vortex). Solution should become blue (if indicator is added). | + | *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. | + | *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. | + | *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. | + | *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. | + | *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. | + | *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. | + | *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. | Store the minipreps at -20. The concentration obtained can be measured by Nanodrop. | ||
Revision as of 21:37, 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.
