User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/06/20

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ABSTRACT

 * Beggining of the protocol for making E.coli BL21 (DE3) competent cells.


 * Today I prepared 1 liter of liquid LB medium, as the liquid LB we have was contaminated. I followed Miguel Ángel's protocol.
 * I put 5 ml of liquid LB with BL21 (DE3) cells at 9:00 am, they were left incubating until 1:30 pm to continue with the protocol for making competent cells. The next step was to put 1 ml from the 5 ml of liquid LB with the bacteria into one big 1 l flask with 250 ml of liquid LB. This was performed twice, so there's 500 ml of liquid medium with BL21 (DE3) cells up to this step. These flasks were left incubating until they had an optical density of 0.4.
 * I searched for papers that described the use of β-galactosidase with the TAT pathway. I found several papers, fusing our export tag to this enzyme, should prove sufficient to readily characterize this tag. We should just perform the periplasm isolation protocol and put this extract into a medium that has the appropiate reactant (bromo-chloro-indolyl-galactopyranoside, aka X-gal); if the tag worked properly, then the medium should be colored blue.


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