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Team:TUDelft/13 August 2009
From 2009.igem.org
Lab Notebook
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13 August 2009
Calin
Knockout day. [http://openwetware.org/wiki/NanoBio:_Protocol_for_gene_knockout NanoBio: Protocol for gene knockout]
Made 1M l-arabinose stock. 150mg into 0.85 mL ddH20.
Digested CE with EcoRI (10uL DNA).
Yesterdays gel:
| Well | Part | Expected Plasmid Size | Status |
| 1 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
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| 17 | trbK X+P | ✔ | |
| 18 | GFP-gen E+S | ✔ | |
| 19 | pTet+RBS E+S | ? | |
| 20 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ |
Made 12 plates. 4 High KAN. 4 Low KAN. 4 TRI.
Placed 50uL of overnight R751 + pKD46 culture into 4 x 5 mL tubes.
In 30C incubator at 11:15
11:45 0.034 OD
12:30 0.065 OD
13:17 0.126 OD
Induced +L-arabinose tubes with L-arabinose (40uL 1M stock added to 4mL). Control tube has no L-arabinose added.
In 30C incubator at 13:25
14:40 0.497 OD
on Ice at 14:57
centrifuge 1 @ 15:13 - 10 min 4000 rcf 3C
centrifuge 2 @ 15:44 - 10 min 4000 rcf 3C
centrifuge 3 @ 16:10 - 10 min 4000 rcf 3C
12 plates made, of which 8 are various controls
| Plate ID | Knockout | Variables | AB |
| 1H | oriTR_KO | +L-arabinose +PCR | 1xKAN |
| 1L | oriTR_KO | +L-arabinose +PCR | 0.5xKAN |
| 2 | oriTR_KO | +L-arabinose -PCR | TRI |
| 3H | oriTR_KO | -L-arabinose +PCR | 1xKAN |
| 3L | oriTR_KO | -L-arabinose +PCR | 0.5xKAN |
| 4 | oriTR_KO | -L-arabinose -PCR | TRI |
| 5H | trbK_KO | +L-arabinose +PCR | 1xKAN |
| 5L | trbK_KO | +L-arabinose +PCR | 0.5xKAN |
| 6 | trbK_KO | +L-arabinose -PCR | TRI |
| 7H | trbK_KO | -L-arabinose +PCR | 1xKAN |
| 7L | trbK_KO | -L-arabinose +PCR | 0.5xKAN |
| 8 | trbK_KO | -L-arabinose -PCR | TRI |
Made 5mL tube cultures for assemblies CB and CC.
