I could use some advice.
I've done three transformations now and I think my ligation AND insertion rates are pretty low. I think my bacteria that carry the plasmid are getting out-competed so I made liquid Ampicilin cultures and incubated them overnight to remove any that don't carry my plasmid.
I dilute the culture 1:1000 and plate 3uL to get a good distribution of colonies. About 5-10% are white, the rest are blue of course. The white colonies all carry the plasmid, but no insert. I ran a colony PCR on 8 samples from 4 plates and all 32 tubes had the plasmid but no insert.
Tomorrow I'm considering making 3-4 plates so I have enough white colonies to fill a 96 tube rack.
My other idea is that if my ligation efficiency is low, I'm incubating too long and may try a 2-4 hour incubation period instead of 12-16.
Any advice or ideas?