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.
 * 3) *Ligations:
 * 4) *# 'J04500+KaiA+J04500+KaiC'+'J04500+KaiB'
 * 5) *# 'Promoter(rbs)+'KaiA'
 * 6) *# 'B0015'+'J04500+KaiB'
 * 7) *# 'B0015'+'J04500+KaiC'
 * 8) *# 'B0015+J04500+KaiB'+'B0015+J04500+KaiC'
 * 9) *# 'Promoter(rbs)+KaiA'+'B0015+J04500+KaiB+B0015+J04500+KaiC'
 * 10) *Progression
 * 11) ** Step 1: Midiprep
 * 12) **# B0015
 * 13) **# J04500+KaiB
 * 14) **# J04500+KaiC
 * 15) **# J04500+KaiA+J04500+KaiC
 * 16) ** Step 2: Digestion
 * 17) **# B0015/SP important
 * 18) **# J04500+KaiB/XP important
 * 19) **# J04500+KaiC/XP
 * 20) **# J04500+KaiA+J04500+KaiC/SP
 * 21) ** Step 3: CIP/Gel Purification
 * 22) ** Step 4: Ligation/Transformation
 * 23) **# 'J04500+KaiA+J04500+KaiC'+'J04500+KaiB'
 * 24) **# 'B0015'+'J04500+KaiB'
 * 25) **# 'B0015'+'J04500+KaiC'
 * 26) ** Step 5: Midiprep
 * 27) **# KaiA
 * 28) **# Promoter(rbs)
 * 29) **# B0015+J04500+KaiB
 * 30) **# B0015+J04500+KaiC
 * 31) ** Step 6: Digestion
 * 32) **# KaiA/XP
 * 33) **# Promoter(rbs)/SP
 * 34) **# B0015+J04500+KaiB/XP
 * 35) **# B0015+J04500+KaiC/XP
 * 36) ** Step 7: CIP/Gel Purification
 * 37) ** Step 8: Ligation/Transformation
 * 38) **# 'Promoter(rbs)+'KaiA'
 * 39) **# 'B0015+J04500+KaiB'+'B0015+J04500+KaiC'
 * 40) ** Step 9: Midiprep
 * 41) **# Promoter(rbs)+KaiA
 * 42) **# B0015+J04500+KaiB+B0015+J04500+KaiC
 * 43) ** Step 10: Digestion
 * 44) **# Promoter(rbs)+KaiA/SP
 * 45) **#B0015+J04500+KaiB+B0015+J04500+KaiC/XP
 * 46) ** Step 11: CIP/Gel Purification
 * 47) ** Step 12: Ligation/Transformation
 * 48) *** 'Promoter(rbs)+KaiA'+'B0015+J04500+KaiB/SP+B0015+J04500+KaiC'
 * 49) Once J04500+KaiA+J04500+KaiC+J04500+KaiB is complete, borrow a chemostat (or build one? =\).
 * 50) * 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

Estimated time: 170 minutes/ 2h 50m

Adapted from the QIAGEN Midiprep protocol.