getting there

Woohooo! I finally got blue colonies. I was looking through my old plates (about 2 weeks old) so that I can throw away the unnecessary ones. As I got to a set of plates from August 8, I saw blue colonies. That was VERY surprising! I looked back in my lab notebook and my comment was ‘No blue colonies (’. So how come there are blue colonies on the same plates now? The blue colonies are also very dark blue and definitely seem to be growing fine.

Without hesitating further, I picked the blue colonies and streak them on LB plates supplemented with ampicillin, IPTG and X-Gal. There were 8 of them. The next day, I found that only 6 of them were still blue. The other two had reverted to white colonies. I am not sure what they mean but it’s an indication that these clones are not very stable. Delighted to see that these blue colonies are indeed expressing the lacZ gene, I inoculated them for plasmid prep. Isolated the plasmids yesterday and digested them today. Just before I left, I managed to analyzed the bands from these digestion and guess what…they ARE DEFINITELY different from my vector and from the plasmid that my gene came from. This is good news! It does mean success yet but definitely a step forward. At least I have something to troubleshoot on now. I was about to give up as I was repeating the same protocols again and again, changing one factor at one time.

Came back here after dinner to look at the restriction digestion patterns of the plasmids. They are close to the prediction based on my calculations but something is still not right. Will be digesting with different enzymes tomorrow to get more patterns. Will also have to start writing up a new protocol to confirm the expression of the gene. Woohoooo!! Maybe I can finally give Tom (my boss) the green light to buy the fluorometer.