User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/05/24

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χρόνος πέρασμα May 24th 2011

  • After some labmates did all the work pertinent with the transformation, I had to make the petri dishes and inoculate the E.coli DH5α with all the variants.
  • The variants were:

a) The digested plasmid, blunted and with the PCR amplification product, all this treated with ligase. Expected outcome: Red colonies; this means the E.coli that did grew contain a plasmid that confers gentamicin resistance (pBBRMCS5 backbone) and the amplification product that has the RFP gene.

b) The digested plasmid, blunted and dephosphorilated, all this treated with ligase. Expected outcome: No colonies; even when it was treated with ligase, there shouldn't be any circularized plasmid as two DNA tips can't be joined if they are dephosphorilated. So no gentamicin resistance gene is available for the bacteria to survive.

c) The digested plasmid and blunted, all this treated with ligase. Expected outcome: Grey normal colonies, the pBBRMCS5 plasmid, without the RFP gene, should be recircularized, each bacteria containing this plasmid should survive.

d) An empty petri dish. Expected outcome: No colonies; as the solid medium was added with gentamicin, no bacteria should be able to survive, unless they possess some sort of resistance to the antibiotic.

  • I used an already made solid medium LB that was stored in the lab. Added 15 µl of gentamicin (concentration 10 µl gentamicin/1ml) to 150ml of LB. Let the LB dry in the petry dishes and started plating the bacteria when the solid LB medium tore apart... I let the other dishes dry for another 15 minutes and tried again, they tore apart again. The conclusion was that the LB medium hadn't been correctly prepared.
  • I followed the protocol that Miguel gave to us. 1 liter. The problem was that I didn't know the agar should be disolved first, before adding it to the other components... this must be done to make it easier to disolve all the components in 1 liter of destilled water. I moved the solution to two 500ml flasks, and put them into the autoclave machine to sterilize them.



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