Team:TUDelft/27 July 2009

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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