Team:Aachen/Wetlab/Protocol16

Cre-LOXp Treatment of yeast cells:

Material:


  • YPD+hygromycin (conc. 250 mg/L) plates.

  • YPD plates.

  • auxotrophie-media-plates of the auxotrophie you want to remove.

  • liquid induce-media, with the GAL1-Promotor 100%Galactose YPD-Media.

  • plasmid containing cre- protein and an inducible promoter upstream (e.g. pSH69 of E.coli Stammnummer 2757 (iAMB)).


Procedure:


  1. Transformation of the cells that have to be treated with the plasmid that contains the gene for the cre-protein. For example the plasmid pSH69 from E.coli Stammnummer 2757 (iAMB). The protocol will be described in usage of this plasmid, which contains a GAL1 Promoter upstream of the cre-protein-DNA and a hygromycin Marker Gene.

  2. The Transformants get plated on YPD + hygromycin (conc. 250 mg/L) plates.

  3. When the colonies on the plate grew, use the method "Abschwämmen" to dilute the Colonies on Medium. That means you pour some induce- media onto the plate, so all colonies are covered with media. Than you remove all the colonies from the plate into the media with a drigalski-spatel or something else.

  4. .Take the media from the plates with all the cells from the colonies in it and pour it into a induce- media, in this case Galactose+YPD -media (because of the GAL1 Promoter).

  5. Leave the cells in the induce- media for 4-5 h at 28-30 ℃. Now the cre-protein gets expressed and it deletes all the DNA sequences which are between LOXp- sites.

  6. Plate the cells on a YPD- plate but before prepare a dilution-row (Verduennungsreihe) of the cells in the induce media to get single colonies on the plate.

  7. After grwoth of colonies on the YPD-plate, check 5 colonies or more for success of the cre treatment: You mark 5 colonies or more on the YPD- plate and take half of each to spread them on auxotrophie-media-plates of exactly the auxotrophie you want to remove from your cells. Only if the cells can NOT grow on the auxotrophie-media-plate, the cre-treatment was successful!