Team:Bordeaux/First

Day 1 : 03-07-2012

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For the first week of our project lab we will extract the biobricks needed for the first operon construction.

On the first Day we did a transformation by electroportation for each part needed.
We put 50 µL of DH5α competent cells into 2ml tube. Added 1,5 µL of the resuspended DNA to the 2ml tube.
We then transfered the cells + DNA into pre-chilled electroporation cuvettes.
After the electroporation we added 200 μl of SOB media and incubated the cells at 37ºC for 45 min.
We plated 50 µl of the transformation onto LB dishes,

with Kanamicin 50mg/mL Antibiotic for 1.A and 1.E and Ampicilin 100 mg/mL for 1.B 1.C 1.D

We then put the plate to incubate at 37ºC for 16 hours.

Day 2 : 04-07-2012

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The incubated cells grown well overnight giving a lot of colonies except for 1.E witch gave only one colony
We picked single colonies and put them in 10 ml LB with ,

Kanamicin 50mg/mL Antibiotic for 1.A and 1.E and Ampicilin 100 mg/mL for 1.B 1.C 1.D

Then incubated and agitated the tubes at 37ºC for 22 hours
We did a Transformation for the RBS and terminator needed for our constructions

We followed the same transformation protocol as Day 1
And plated 100μL of the transformants (not 50μL regarding the poor results of the plate 1.E ) in LB + ampicilin 100mg/mL
We then incubated the two plates 37ºC for 19 hours

Day 3 : 05-07-2012

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The two plates 5.A and 5.B gave a good amount of colonies but 100μL is too much to inoculate
We picked single colonies and put them in 10 ml LB with Ampicilin 100mg/mL And put the tube to grow at 30°C overnight
The tube cultures seem to have gone well but again 1.E is slightly clearer than the others
We precipitated tree 2mL tubes for each Biobrick by centrifugation 3min at 13000rpm
We then made a miniprep for one of the tubes and froze the two tubes left
We used a QIAprep® Spin Miniprep kit from QIAGEN® Read Protocol

Day 4 : 06-07-2012

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The two tubes put to incubate overnight grown a good amount of bacteria
We did a centrifugation for tree 2 ml tubes per biobrick 3 min at 13 000 rpm
We froze 4 of the tubes and did a miniprep for each tube left.
We used as yesterday a QIAprep® Spin Miniprep kit from QIAGEN® Read Protocol
We then made a titration of the samples with the nanodrop Read Procedure
Only 1.A gave average results with 28 ng/μL the others < 20 ng/μL
We suspect the problem come from the competent cells we used because we used a different stock for 1.A and 1.E1.E giving bad results from the beginning it might come from a bad transformation .
We will verify this theory next week by changing our competent cells .
Next week we will also extract the plasmids needed for the construction of the third operon because we are still waiting the requested biobricks (BBa_K091106 , BBa_K091001, BBa_K091002) to be delivered to construct our second an fourth operon.