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Team:TUDelft/27 July 2009
From 2009.igem.org
Lab Notebook
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27 July 2009
Calin
Placed GFP / RFP plates in fridge. Ordered some trimethoprim. Placed pSB1AK3 cells in tube for culturing. Finished writing Matlab code to plot parameter space. Cleared up confusing equations for mRNA and protein.
Sriram
Got two colonies in the RBS[B0034] and pLacI[R0010] plates kept in the weekend. Hence it is confirmed that the transformation efficiency was not that good for the newly prepared competent cells.
Prepared 1 litre of LB-Agar and separated in 4x500 ml beakers and sent for autoclaving with a beaker of eppendorfs. Since it was not received before 18:00, we couldn't perform transformation for the the 6 plasmid backbone biobricks today and hence just diluted the frozen DNA of them. These biobricks will be used in all the modules of this project Looked into the protocol that will be used for transformation of them in ccdB resistant competent cells.
Ran the agarose gel electrophoresis with the 10 biobricks [R0040, J23008, J23031, B0015, K081013, S03335, S03473, I714031, E0840, J23100] for 1 hour to check the extracted DNA. It must be continued tomorrow hence the gel was preserved in fridge.
Inoculated the top10 cells for preparing competent cells, λp-GFP [R0040] and one of the two colonies got from RBS[B0034] and pLacI[R0010] plates, in 5 ml culture tubes for plasmid DNA extraction.
Orr
Used mfold to produce 3 locks: one with weak RBS, one with weaker RBS and one with strong RBS.
Using the 2-state hybridization server, combined the key3c with each of the 4 locks (lock3c, weak lock, weaker lock and strong lock) to obtain the hybridised structure.
Created weak key, weaker key and strong key sequences for each of the last three locks by taking the hybridization of each lock with the key3c and correcting the kinks by changing the key3c sequence.
Biobricked the weak, weaker and strong keys into the registry.
Tim Weenink
Nanodropped the plasmid isolations I did on friday:
The *I is the code for the biobrick I have constructed, that contains the I-SceI cleavage site. The !A is an abbreviation for a brick containing RBS32 + GFP-LVA.
| Part | DNA concentration in ng/µl | 260/280 | 260/230 |
| *I1 | 88.9 | 1.96 | 2.19 |
| *I2 | 80.3 | 1.95 | 2.20 |
| *I3 | 78.8 | 1.96 | 2.34 |
| *I5 | 78.6 | 2.03 | 2.37 |
| *I6 | 93.7 | 1.98 | 2.29 |
| *I7 | 82.3 | 1.94 | 2.20 |
| BBa_K142205 | 73.0 | 1.98 | 2.28 |
| !A | 15.3 | 1.97 | 1.67 |
I also did some restrictions (all in µl) (incubated for 35min at 37ºC, then inactivated):
| Ingredient | *I3 | *I6 | Control plasmid |
| I-SceI enzyme | 0.2 | 0.2 | 0.2 |
| DNA | 6.25 | 6.25 | 1.0 |
| I-SceI buffer | 2.5 | 2.5 | 2.5 |
| BSA | 0.25 | 0.25 | 0.25 |
| H2O | 15.80 | 15.80 | 20,80 |
| Total | 25.0 | 25.0 | 24.75 |
And some more (10min 37ºC, 20min inactivation):
| Ingredient | *S | !A |
| EcoRI-HF enzyme | 0.5 | 0.5 |
| PstI enzyme | 0.5 | 0.5 |
| DNA | 3.5 | 15.0 |
| NEB2 buffer | 2.5 | 2.5 |
| BSA | 0.25 | 0.25 |
| H2O | 17.75 | 6.2 |
| Total | 25.0 | 24.95 |
Then loaded the gel:
| Well | Part | µl of sample | µl of loading buffer |
| 1 | *I3 unrestricted | 1 | 2 |
| 2 | *I3 restricted | 5 | 2 |
| 3 | *I6 unrestricted | 1 | 2 |
| 4 | *I6 restricted | 5 | 2 |
| 5 | restricted control plasmid | 5 | 2 |
| 6 | *S unrestricted | 1 | 2 |
| 7 | *S restricted | 5 | 2 |
| 8 | !A unrestricted | 1 | 2 |
| 9 | !A restricted | 5 | 2 |
| 10 | Smartladder | 6 | 0 |
