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Team:TUDelft/24 August 2009
Lab Notebook
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24 August 2009
Sriram
Today I did the restriction of all the DNA I minipreped on friday. The gel image below shows the restricted samples.
Then I also ligated and transformed the assemblies for riboregulator [5. pTet + Lock3c (Chloramphenicol backbone), 6. cI + Double Terminator (Chloramphenicol backbone), 7. λp-in + RBS-GFP-Double Term (Chloramphenicol backbone), 8. pLacI + key3c (Amp-Kan backbone)]and stopped the negative cascade assembly since Daniel's backup plan for it is progressing very well.
Since Daniel needed the λp-GFP (BBa_S03335) for his assembly in Chloramphenicol backbone, I did that assembly as well.
Daniel
Transformaltion of friday 21-08-2009 looked good. On each plate there were 80-90 colonies
Digestion of assemblies in order to do the second round of assembly. Gel 1% (left) for Calin´s PCR and 2% (right) for my digestions and Calin´s PCR
Left Gel (2%)
| Well | Assembly | Expected Plasmid Size | Status |
| 1 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 2 | col PCR 1 | 1616 | ✖ |
| 3 | col PCR 2 | 1616 | ✖ |
| 4 | col PCR 3 | 1616 | ✖ |
| 5 | col PCR 4 | 1616 | ✖ |
| 6 | col PCR 5 | 1616 | ✖ |
| 7 | col PCR 6 | 1616 | ✖ |
| 8 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ |
Right Gel (1%)
| Well | Assembly | Expected Plasmid Size | Status |
| 1 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ✔ | |
| 2 | 4A | 824 | ? |
| 3 | 5A | 1040 | ✔ |
| 4 | 2A | 214 | ? |
| 5 | 3A | 183 | ? |
| 6 | 1A | 1076 | ? |
| 7 | col PCR 1 | 1616 | ✖ |
| 8 | col PCR 2 | 1616 | ✖ |
| 9 | col PCR 3 | 1616 | ✖ |
| 10 | col PCR 4 | 1616 | ✖ |
| 11 | col PCR 5 | 1616 | ✖ |
| 12 | col PCR 6 | 1616 | ✖ |
Prepare IPTG 1M for further induction. I tried to do an experiment with 1A which is GFP under control of PLacI however the culture glows even though there is no IPTG induction.
We got the oligo for the two locks and keys I designed.
