User:Hetmann/Notebook/2006-11-17
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Updated Goals for November/December
- Relabel and organize tubes within BL5088 and BL5096.
- Starting the week of Monday, 11/13, we are shooting for a goal of one successful ligation per week. Ligations of all constructs should be complete before Christmas break.
- Ligations:
- 'J04500+KaiA+J04500+KaiC'+'J04500+KaiB'
- 'Promoter(rbs)+'KaiA'
- 'B0015'+'J04500+KaiB'
- 'B0015'+'J04500+KaiC'
- 'B0015+J04500+KaiB'+'B0015+J04500+KaiC'
- 'Promoter(rbs)+KaiA'+'B0015+J04500+KaiB+B0015+J04500+KaiC'
- Progression
- Step 1: Midiprep
- B0015
- J04500+KaiB
- J04500+KaiC
- J04500+KaiA+J04500+KaiC
- Step 2: Digestion
- B0015/SP important
- J04500+KaiB/XP important
- J04500+KaiC/XP
- J04500+KaiA+J04500+KaiC/SP
- Step 3: CIP/Gel Purification
- Step 4: Ligation/Transformation
- 'J04500+KaiA+J04500+KaiC'+'J04500+KaiB'
- 'B0015'+'J04500+KaiB'
- 'B0015'+'J04500+KaiC'
- Step 5: Midiprep
- KaiA
- Promoter(rbs)
- B0015+J04500+KaiB
- B0015+J04500+KaiC
- Step 6: Digestion
- KaiA/XP
- Promoter(rbs)/SP
- B0015+J04500+KaiB/XP
- B0015+J04500+KaiC/XP
- Step 7: CIP/Gel Purification
- Step 8: Ligation/Transformation
- 'Promoter(rbs)+'KaiA'
- 'B0015+J04500+KaiB'+'B0015+J04500+KaiC'
- Step 9: Midiprep
- Promoter(rbs)+KaiA
- B0015+J04500+KaiB+B0015+J04500+KaiC
- Step 10: Digestion
- Promoter(rbs)+KaiA/SP
- B0015+J04500+KaiB+B0015+J04500+KaiC/XP
- Step 11: CIP/Gel Purification
- Step 12: Ligation/Transformation
- 'Promoter(rbs)+KaiA'+'B0015+J04500+KaiB/SP+B0015+J04500+KaiC'
- Step 1: Midiprep
- Ligations:
- Once J04500+KaiA+J04500+KaiC+J04500+KaiB is complete, borrow a chemostat (or build one? =\).
- Test the construct, using Western Blot analysis.
Efficient Midiprep Protocol (for HC plasmid)
Pre-Midiprep
Put buffer P3 in the refrigerator. Check buffer P2 for precipitate; if there is then redissolve @ 37°C.
Midiprep
Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. Big centrifuge: SLA-1500; Rotor code 28; 6290 rpm. Big centrifuge: HB6; Rotor code 23; 6060 rpm.
- Alternatively, centrifuge cultures in 50 mL falcon tubes for 15 minutes at 4400 rmp in room 5096, and dump supernatant. Simultaneously, turn on the big centrifuge in room 5088, fill a bucket of ice, and place the medium rotor into the big centrifuge.
- Medium rotor: SS34, rotor code 05.
- Resuspend the bacterial pellet in 4 ml Buffer P1.
- Add 4 ml Buffer P2, mix by inverting 4-6 times, then incubate at room temperature for 4 minutes.
- Add 4 ml Buffer P3, mix by inverting 4-6 times, then incubate on ice for 15 minutes.
- Mix the sample again.
- Centrifuge at 13,000 rpm for 30 min at 4°C, and remove the supernatant promptly. Concurrently, equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow the column to empty by gravity flow.
- Centrifuge in non-glass tube. '<~ this should be all of the tubes present in lab'
- Centrifuge the supernatant again at 13,000 x g for 5 min at 4°C, and remove the supernatant promptly.
- Apply supernatant to QIAGEN-tip and allow it to enter the resin by gravity flow.
- Wash the QIAGEN-tip twice with 10 ml Buffer QC, and allow to move through the QIAGEN-tip by gravity flow.
- Elute DNA with 5 ml QF.
- Collect the eluate in a 10 ml tube.
- Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA.
- Mix and centrifuge immediately at 11210 rpm for 30 minutes at 4°C, and carefully decant the supernatant. Then make sure all of the isopropanol is out.
- Wash DNA pellet with 2 ml of room-temperature 70% ethanol and centrifuge at 11210 rpm for 10 minutes.
- Carefully decant the supernatant, and don't disturb the pellet - nevertheless, make sure that no ethanol remains.
- Air-dry the pellet for 5-10 min, and redissolve the DNA in 250 ul ddH2O
- Alternatively, centrifuge cultures in 50 mL falcon tubes for 15 minutes at 4400 rmp in room 5096, and dump supernatant. Simultaneously, turn on the big centrifuge in room 5088, fill a bucket of ice, and place the medium rotor into the big centrifuge.
Estimated time: 170 minutes/ 2h 50m
Adapted from the QIAGEN Midiprep protocol.