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Team:TUDelft/30 July 2009
From 2009.igem.org
Lab Notebook
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30 July 2009
Tim Weenink
prepared assembly mixes for !B, !C and !D constructs. Standard Ginko assembly kit was used. DNA concentrations as below (these mixtures were added to 7.5 µl of digestion mixture):
| Assembly name | position | Components | volume in µl |
| !B | Upstream | !A DNA | 33.0 |
| H20 | 9.5 | ||
| Downstream | BBa_B0015 DNA | 10.0 | |
| H20 | 32.5 | ||
| Backbone | pSB1AC3 DNA | 6.5 | |
| H20 | 36 | ||
| !C | Upstream | BBa_R0040 DNA | 10.0 |
| H20 | 32.5 | ||
| Downstream | *I6 DNA | 5.5 | |
| H20 | 37.0 | ||
| Backbone | pSB1AC3 DNA | 6.5 | |
| H20 | 36 | ||
| !D | Upstream | *I6 DNA | 5.5 |
| H20 | 37.0 | ||
| Downstream | BBa_K145201 DNA | 18.0 | |
| H20 | 24.5 | ||
| Backbone | pSB1AC3 DNA | 6.5 | |
| H20 | 36 |
Ligated and transformed assemblies according to protocol.
15:20 Done plating the assemblies.
15:40 inoculated *S and *T cultures, four colonies each
16:55 Inoculated !A cultures from glycerole stock. Two tubes.
Calin
Plated three backbones left over from the transformation the other day. Did a digest on oriT-R with E and P. Made 3 x 5ml culture tubes for the pSB4C5 backbone which had a few colonies. Made electrocompetent cells with the [http://openwetware.org/wiki/Knight:Preparing_electrocompetent_cells Knight:Preparing electrocompetent cells] protocol. Flash froze tube in liquid nitrogen. Checked backbone concentrations:
| Part | Concentration (ng/uL) |
| rbs-GFP-term I | 102.6 |
| rbs-GFP-term II | 113.5 |
| pSB1A3 I | 89.1 |
| pSB1A3 II | 83.2 |
| pSB1A3 III | 91.9 |
| pSB1A3 IV | 84.7 |
| pSB4A5 I | 77.7 |
| pSB4A5 II | 83.9 |
| pSB4A5 III | 99.0 |
| pSB4A5 IV | 116.1 |
| pSB1C3 I | 102.3 |
| pSB1C3 II | 49.4 |
| pSB1C3 III | 81.5 |
| pSB1C3 IV | 66.0 |
Ran gel on backbones and oriT digest. Gel left for too long.
| Well | Part | Expected Plasmid Size | Status |
| 1 | rbs-GFP-term I | 2957 | ✔ |
| 2 | rbs-GFP-term II | 2957 | ✔ |
| 3 | pSB1A3 I | 2832 | ✔ |
| 4 | pSB1A3 II | 2832 | ✔ |
| 5 | pSB1A3 III | 2832 | ✔ |
| 6 | pSB1A3 IV | 2832 | ✔ |
| 7 | pSB4A5 I | 4070 | ✖ |
| 8 | pSB4A5 II | 4070 | ✖ |
| 9 | pSB4A5 III | 4070 | ✖ |
| 10 | pSB4A5 IV | 4070 | ✖ |
| 11 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 12 | pSB1C3 I | 2747 | ✔ |
| 13 | pSB1C3 II | 2747 | ✔ |
| 14 | pSB1C3 III | 2747 | ✔ |
| 15 | pSB1C3 IV | 2747 | ✔ |
| 16 | oriT E + P digest | 2079 + ~278 | ? |
| 17 | oriT E + P digest | 2079 + ~278 | ? |
| 18 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ |
Tim Vos
Minipreped 4 x pSB1C3, 4 x pSB4A5, 2 x rbs-GFP, and 4 x pSB1A3. One pSB4A5 tube is incorrect, it shows RFP expression. Also loaded the gel.
Orr
Made some chemically competent cells using the TSS buffer protocol. Started making a Java program that will allow lock sequence design based on the chosen RBS, and will design the equivalent key based on the lock sequence. The program will incorporate a database with all the working RBS segments in the iGEM registry, so that the user can choose which RBS he wants to use.
