I already have performed the blue white screening and got the white colonies (transformants). I cultured the transformants onto blue white screening plate again (already containing X-gal, ampicillin, and IPTG). In some days, I found my transformant colonies turn into blue colonies. Why could this happen? Is this false positive?
Yes, it happen as transformants often loose the plasmid over time. The recommended way to keep them for long period is on Glycerol.
well after overnight incubation (at FIRST experiment) you have to keep your plates at 4c for some hours to get the TRUE white color colonies. some times at first you see colonies are white but after some time turn to blue, so would suggest to incubate at 4c following 37c overnight incubation then to choose the colony.
- Depends upon the insert stability in your cloned plasmid. A cloned plasmid may loose the insert after a period of time which results in the blue coloration of your colonies. There are some high-efficiency competent cells available commercially which provide high insert stability due to certian mutations in the cells.
- The best way to preserve a cloned plasmid is by preparation of glycerol stock, otherwise prolonged incubation at 4 degrees may lead to insert loss.
- Secondly, as suggested by Saber, after your colonies appear, to further enhance the blue coloration and to distinguish between blue and white, keep your plate at 4 degrees for 24-48hrs. Incubation at lesser degree enhances the blue coloration
- Badges
- Science method