User:Hetmann/Notebook/2006-11-17

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Updated Goals for November/December

1. Relabel and organize tubes within BL5088 and BL5096.
2. 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:
     1. 'J04500+KaiA+J04500+KaiC'+'J04500+KaiB'
     2. 'Promoter(rbs)+'KaiA'
     3. 'B0015'+'J04500+KaiB'
     4. 'B0015'+'J04500+KaiC'
     5. 'B0015+J04500+KaiB'+'B0015+J04500+KaiC'
     6. 'Promoter(rbs)+KaiA'+'B0015+J04500+KaiB+B0015+J04500+KaiC'
   • Progression
     • Step 1: Midiprep
       1. B0015
       2. J04500+KaiB
       3. J04500+KaiC
       4. J04500+KaiA+J04500+KaiC
     • Step 2: Digestion
       1. B0015/SP important
       2. J04500+KaiB/XP important
       3. J04500+KaiC/XP
       4. J04500+KaiA+J04500+KaiC/SP
     • Step 3: CIP/Gel Purification
     • Step 4: Ligation/Transformation
       1. 'J04500+KaiA+J04500+KaiC'+'J04500+KaiB'
       2. 'B0015'+'J04500+KaiB'
       3. 'B0015'+'J04500+KaiC'
     • Step 5: Midiprep
       1. KaiA
       2. Promoter(rbs)
       3. B0015+J04500+KaiB
       4. B0015+J04500+KaiC
     • Step 6: Digestion
       1. KaiA/XP
       2. Promoter(rbs)/SP
       3. B0015+J04500+KaiB/XP
       4. B0015+J04500+KaiC/XP
     • Step 7: CIP/Gel Purification
     • Step 8: Ligation/Transformation
       1. 'Promoter(rbs)+'KaiA'
       2. 'B0015+J04500+KaiB'+'B0015+J04500+KaiC'
     • Step 9: Midiprep
       1. Promoter(rbs)+KaiA
       2. B0015+J04500+KaiB+B0015+J04500+KaiC
     • Step 10: Digestion
       1. Promoter(rbs)+KaiA/SP
       2. 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'
3. 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.

1. 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.
2. Resuspend the bacterial pellet in 4 ml Buffer P1.
3. Add 4 ml Buffer P2, mix by inverting 4-6 times, then incubate at room temperature for 4 minutes.
4. Add 4 ml Buffer P3, mix by inverting 4-6 times, then incubate on ice for 15 minutes.
5. Mix the sample again.
6. 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'
7. Centrifuge the supernatant again at 13,000 x g for 5 min at 4°C, and remove the supernatant promptly.
8. Apply supernatant to QIAGEN-tip and allow it to enter the resin by gravity flow.
9. Wash the QIAGEN-tip twice with 10 ml Buffer QC, and allow to move through the QIAGEN-tip by gravity flow.
10. Elute DNA with 5 ml QF.
11. Collect the eluate in a 10 ml tube.
12. Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA.
13. 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.
14. Wash DNA pellet with 2 ml of room-temperature 70% ethanol and centrifuge at 11210 rpm for 10 minutes.
15. Carefully decant the supernatant, and don't disturb the pellet - nevertheless, make sure that no ethanol remains.
16. Air-dry the pellet for 5-10 min, and redissolve the DNA in 250 ul ddH2O

Estimated time: 170 minutes/ 2h 50m

Adapted from the QIAGEN Midiprep protocol.