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Team:TUDelft/11 August 2009
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imaged the gel i ran yesterday: | imaged the gel i ran yesterday: | ||
| - | [[Image:Tim110809!B!C!S.png|thumb|420px]] | + | [[Image:Tim110809!B!C!S.png|thumb|center|420px]] |
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. | 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. | ||
Revision as of 08:13, 20 October 2009
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 | !E Upsteam | 1114 | ✖ |
| 2 | !F Upsteam | 913 | ✖ |
| 3 | !F downstream | 919 | ✔ |
| 4 | Backbone pSB1AK3 | ✔ | |
| 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 | ✔ |
Daniel
First success, got colonies in all the plates which is strange due everybody had difficulties with heat shock method which made us decide to make more electro-competent cells. Me and Sriram prepare these cells today.
From the colonies in the plates I made tube cultures by duplicate, let's wait one night for more growth.
