X-Gal Filter Assay
1) Cut nitrocellose membrane and chromatography paper to the size of a petri dish. Label the nitrocellulose to mark the orientation.
2) Obtain liquid nitrogen.
3) Make X-Gal solution. Add 1mg/ml X-Gal to 5ml of Z-buffer. (The stock solutions of X-Gal in the -20ºC freezer are 50mg/ml so add 100ul to 5ml Z-buffer).
4) Place the chromatography paper in a petri dish and soak it with ~3ml X-Gal solution.
5) Lay the nitrocellulose onto the plate of yeast and allow it to wet completely.
6) Lift the nitrocellulose off of the plate carefully to avoid smearing the colonies.
7) Place the nitrocellulose in the liquid nitrogen to permeabilize the cells. 10-15 seconds is sufficient. The nitrocellulose may either be submerged or placed on a foil float.
8) Carefully remove the nitrocellulose from the liquid nitrogen (it will be brittle). Place the nitrocellulose cell-side up on the chromatography paper in the petri dish. Allow it to wet completely.
9) Wrap the petri dish in foil and incubate at 30ºC for minutes to overnight.
10) Putative positives can be picked directly off of the nitrocellulose filter if streaked immediately. All putative positives should be tested a second time to confirm.
Z-Buffer (1L)
Na2HPO4 7H2O 16.1g
NaH2PO4 H2O 5.5g
KCl 0.75g
MgSO4 7H2O 0.25g
2-mercaptoethanol 2.7ml
Adjust pH to 7.0
