Team:Mexico-UNAM-CINVESTAV/Notebook/Week Five

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Week #5

04th October - 08 October 2010

Monday

  • Let's picked up colonys our transformant cells and prepare all to do miniprep.

For this we used 30ml de LB liquid agar medium per falcon tube at 80μ/ml

Cloramphenicol concentration we left overnight culture to 37º


Labo 271.JPG

DSC02107.JPG


Tuesday

With a overnight inoculum we did a miniprep.

The miniprep was done with a Quiagen Kit.

Then we going to do a duble digest with EcoRI and PstI to check out our ligations.


DNA 5μl
Buffer NB2 1μl
EcoRI 0.5μl
PstI 0.5μl
H2O 2.7μl
BSA 0.3μl
Total 10μl


Wensday

We run a gel to confirm our ligations as follow

  • PCR 1 Lig S with Psb1C3 Confirmed
  • PCR 2 Lig S with Psb1C3 Unconfirmed
  • PCR 3 Lig S with Psb1C3 Confirmed
  • PCR 4 Lig C with Psb1C3 Unconfirmed


Conf3.gif
Right

We prepare all to repeat ligation; PCR2 with Psb1C3 and PCR4 with Psb1C3


Thursday

We did ligations (Fast ligation one hour) Pcr 2 with Psb1C3 and PCR 4 with Psb1C3 then transformed DH5α

competent cells, and incubated at 37º overnight.

We ran a gel to test our biobricks for characterisation with the follow results

  • I0260 Positive Plate 2010
  • E0240 Positive Plate 2010
  • R010 Positive Plate 2010
  • J13002 Negative Plate 2007


Example.jpg