5 inserts, 1 colony..dreaming of a better cloning world - (Aug/26/2008 )

Uhm I just found a colony in one of my five agar plates. I will pick it and do a miniprep. This is from the transformation that I had frozen and replated yesteday. The first time I did it I saw one colony in another plate but I thought it had gone wrong because there were none in the other plates.

Now im thinking, if I had picked that one and minipreped it maybe it would have been the right sequence and maybe this one is too. Uhm I would have had two plasmids containing siRNA against my target............

Am I being too hopeful here?

I am picking that damn colony!! Maybe it is the one plasmid that will knock down my gene succesfully lol

May the Goddess of Ligation be on your side. And Lady Luck smile on you!

(But I will do the ligation again. )

I usually keep the leftover of the transformation in the fridge until the next morning If I have too few colonies I spin quickly the leftover of the culture and resuspend in fresh medium ( at higher concentration) before to plate it on new LB plates

I re-transformed the ligations. Since I only had like 6 microlitres left I only used 25 microlitres of my E.coli. I made sure the medium was at least room temperature, I also prewarmed the agar plates and voila!!

I have colonies on three plates

and I can see a little one on other two


!!!!!!!!!!!


yay!!!!!!!!!

Also in an ironic turn of events, the one colony I had after replating the transformation was incorrectly cultured (by me ) in medium without the antibiotic. I realized this today and hurriedly added some for 2 hours but my dream of the one colony being THE ONE seems to have died lol I mean, I cant really use it now if I didnt select properly right??

Thank god I redid the trafo and got colonies which are going to be correctly minicultured. Otherwise this would have been very disturbing.

Also in an ironic turn of events, the one colony I had after replating the transformation was incorrectly cultured (by me ) in medium without the antibiotic. I realized this today and hurriedly added some for 2 hours but my dream of the one colony being THE ONE seems to have died lol I mean, I cant really use it now if I didnt select properly right??

Thank god I redid the trafo and got colonies which are going to be correctly minicultured. Otherwise this would have been very disturbing.

yikes! one learns a lot in this forum, thank you!!