Practical Considerations
The correct type of vector and competent cells are important considerations when planning a blue white screen. The plasmid must contain the lacZα, and examples of such plasmids are pUC19 and pBluescript. The E. coli cell should contain the mutant lacZ gene with deleted sequence (i.e. lacZΔM15), and some of the commonly-used cells with such genotype are JM109, DH5α, and XL1-Blue.
It should also be understood that the lac operon is affected by the presence of glucose. Glucose reduces cAMP level, and cAMP is necessary for the formation of the CRP-cAMP complex required for the initiation of transcription of the lac operon. Presence of glucose therefore inactivates the lac operon, and the lac genes will only be turned on when glucose level drops low enough for the CRP-cAMP complex to form. The media used in agar plate therefore should not include glucose.
X-gal is light-sensitive and therefore its solution and plates containing X-gal should be stored in the dark. Isopropyl β-D-1-thiogalactopyranoside (IPTG), which functions as the inducer of the lac operon, may be used in the media to enhance the production of LacZ.
Read more about this topic: Blue White Screen
Famous quotes containing the word practical:
“And so we ask for peace for the gods of our fathers, for the gods of our native land. It is reasonable that whatever each of us worships is really to be considered one and the same. We gaze up at the same stars, the sky covers us all, the same universe compasses us. What does it matter what practical systems we adopt in our search for the truth. Not by one avenue only can we arrive at so tremendous a secret.”
—Quintus Aurelius Symmachus (A.D. c. 340–402)