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Team:TUDelft/11 August 2009
From 2009.igem.org
Lab Notebook
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11 August 2009
Tim Weenink
imaged the gel i ran yesterday:
This is a really ugly gel, because the comb for creating the wells was too long. The bottom of the wells was too thin. Upon removing the comb, some of the bottoms were damaged, making them leak the loaded samples.
The contents of the lanes is as follows:
Calin
Growth in all tubes, miniprep was done.
Growth on all plates.
Checked concentrations from yesterday:
| Part | DNA concentration |
| trbK | 1.9 |
| pSB1A3 | 5.2 |
| pSB4A5 | 5.2 |
| pSB1C3 | 3.5 |
| GFP-gen | 1.9 |
| oriT-R | 4.0 |
Make R751 wild cells electrocompetent using Knight's procedure.
Did E + P digest on trbK. Did ligation for assembly CB (GFP-gen + oriT-R on pSB1C3) and CE (trbK on pSB1A3).
Made 3 new AMP plates for pKD3, pKD4, and pKD46.
Transformed electrocompetent cells with CB (CAM, timeconstant 4.6) and CE (AMP, timeconstant 4.5) assemblies. Both plates and culture tubes were made. Transformed R751 electrocompetent cells with pKD46, timeconstant 4.2 and left plate in 30C.
Ran a trbK digest with X + P, this was loaded into the last lane in todays 8 well 1% gel and extracted.
Miniprep concentrations were checked by Tim:
| Part | DNA concentration | 260/280 | 260/230 |
| EP-1 | 75.7 | 2.0 | 2.1 |
| EP-2 | 103.2 | 1.9 | 1.3 |
| EP-3 | 139.0 | 1.8 | 1.4 |
| EP-4 | 108.0 | 1.9 | 1.3 |
| EP-7 | 73.8 | 1.9 | 1.1 |
| EP-8 | 119.9 | 2.0 | 2.3 |
| pKD3 | 30.4 | 2.2 | 1.3 |
| pKD4 | 36.2 | 2.0 | 1.0 |
| pKD46 | 90.0 | 1.9 | 1.0 |
| RbCl-2 | 92.6 | 2.0 | 1.9 |
| RbCl-4 | 109.6 | 1.9 | 1.4 |
| RbCl-5 | 162.0 | 1.9 | 1.2 |
| RbCl-6 | 79.8 | 2.0 | 1.7 |
| RbCl-7 | 102.7 | 1.9 | 1.4 |
| pTet-RBS | 68.9 | 1.9 | 1.1 |
| CA | 44.6 | 1.9 | 1.1 |
Todays 26 well 1% gel:
| Well | Part | Expected Plasmid Size | Status |
| 1 | EP-1 | 2953 | |
| 2 | EP-2 | 2915 | ✔ |
| 3 | EP-3 | 2292 | ✔ |
| 4 | EP-4 | 2894 | ✔ |
| 5 | EP-7 | 3040 | |
| 6 | EP-8 | 2374 | |
| 7 | RbCl-2 | 2915 | ✔ |
| 8 | RbCl-4 | 2894 | ✔ |
| 9 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 10 | RbCl-5 | 2176 | |
| 11 | RbCl-6 | 2984 | |
| 12 | RbCl-7 | 3040 | ✔ |
| 13 | pKD3 | 2804 | ✔ |
| 14 | pKD4 | 3267 | ✔ |
| 15 | pKD46 | 6329 | ✔ |
| 16 | pTet-RBS | 2153 | ✔ |
| 17 | CA | 4444 | ✔ |
| 18 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ |
Todays 8 well 1% gel:
| Well | Part | Expected Plasmid Size | Status |
| 1 | |||
| 2 | |||
| 3 | |||
| 4 | |||
| 5 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 6 | pTet-RBS E+S Digest | ||
| 7 | trbK X+P Digest | ✔ |
