I'm cloning a PCR product in pGEMTeasy on DH10b electroMAX cells (Invitrogen). The problem is that after transformating my colonies were light blue and not dark blue so I couldn't discriminate between white and blue colonies very well. Any suggestions?
Put your petri dish with the colonies on the fridge (~4degrees Celsius) for a few hours, usually the the blue colonies get dark blue after that and you can easily differentiate them.
how big is your insert? SigmaAldrich states that small inserts sometimes only partially inactivate the ß-galactosidase and this might lead to light blue colonies.
However, easiest way would be to simply do some mini preps.
Put your petri dish with the colonies on the fridge (~4degrees Celsius) for a few hours, usually the the blue colonies get dark blue after that and you can easily differentiate them.
You can re-patch the white looking colonies after 4 C treatment on LB+ Antibiotic + X-gal + IPTG plate again to reconfirm and then do the colony PC to make sure insert(s) are there.
I agree with Beatriz, I normally put the plates at the fridge for an hour after the growth incubation. It makes the blue color intense. But on the other hand, you can those white-blue colonies to new LB+XGAL+IPTG to see if the color changes.
Thank you very much to all. My insert is 2000 bp so I think B-gal is inactivated! I put my dishes in the fridge for a few hours and colonies became dark blue. But I really dont' understand why they appeared light blue... I performed a lot of cloning experiment and this is the first time I observe this phenomenon.
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