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Team:TUDelft/27 July 2009
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.
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 |
