Team:TUDelft/23 July 2009

From 2009.igem.org

Lab Notebook

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23 July 2009

Tim Weenink

Details of the following to be provided tomorrow:

grew up *S in LC

looked at gel

Did !A assembly again with double the amount of backbone (and rest from fridge). Did it with both DH5alpha and homemade top10 chemically competent cells, to compare transformation efficiencies. Also the right concentration of K was used

PCR'd 8 colonies from my transformation plates with VF2 and I-SceI Reverse primers to see if there was incorporation of the I-SceI restriction site

isolated plasmid from *S

Sriram

The agarose gel electrophoresis was run for another 1 hour and 10 minutes at 110 V. Then the gel was immersed in TBE buffer stained with 20µl of Safe Green gel stain for 1 hour and the bands were viewed to check whether we have the right biobricks. By this we confirmed that all the amplified biobricks to be used in Delay were extracted well and they are pure.

Gel22072009-delay-conj.png


Well Part Expected Plasmid Size Status
1 DNA Ladder
2 BBa_C0040 TetR 2739
3 BBa_ E0040 GFP 2799
4 BBa_E1010 mRFPI 5106
5 BBa_I12006 pLambclin 4507
6 BBa_C0051 cI 2829
7 BBa_J23008 key3c 2450
8 BBa_I12006 pLambclin star 4507
9 BBa_B0015 Double Terminator 3318
10 BBa_R0010 pLacI 2279
11 BBa_K081013 RBS-cI-RBS 2898
12 BBa_J23031 lock3c 2398
13 BBa_R0040 pTet 2133
14 BBa_B0034 RBS 2091
15 BBa_I714031 OriT-R 2357
16 BBa_E0840 GFP generator 2957
17 BBa_J23100 strong promoter 2948
18 BBa_I13522 pTet GFP 3016


For preparing glycerol stocks and plasmid DNA extraction the 7 biobrick colonies [R0010, R0040, J23008, J23031, B0034, B0015, K081013] from the LB agar plates and 2 composite biobricks [S03335, S03473] received from iGEM HQ were cultured in 5 ml tubes with 1xAmp. The 7 biobricks were recultured since we anticipate that we may need more DNA for parts like RBS, Double terminator, etc. in more assemblies and also glycerol stocks were not made for these biobricks.

The 2 composite biobricks [S03335, S03473] were also streaked in LB Agar plates with 1xAmp for future use.

Orr

Prepared TBE and LB Agar mediums using the standard protocols.

Looked at the negative feedback Matlab code in an attempt to write a new code that will enable us to change two variables at a time and check a set of values to find the optimal value for each parameter.


Calin

Gel showed that oriT and promoter miniprep didnt go right. Cultured oriT, promoter, GFP gen in 5 mL tubes. Made 2 GFP plates, an RFP plate, and one with both. Plated parts from MIT (BBa_J23015, BBa_J23055). Added composite part with GFP and oriT to sandbox. Acquired some 20% L-arabinose. Looked into the proofreading PCR kit in the lab for possible use in the knockout (Roche Expand High Fidelity PCR System).