Updates: I think this problem is resolved.

I talked to a friend, and he recommended for me to do colony PCR to check for false positives (I should have included a negative control plate). I picked 6 colonies, and 3 of them have the insert I expected.

So I inoculated those 3 colonies in 37 degree Kan LB over night, still no grow. I got frustrated and just streaked those 3 colonies all over the plate, incubate 37 degree over night, washed them off the plates, collected the cells, mini-prepped them. Digested 5ul of the plasmids with enzymes, they seem to be the correct plasmids I was looking for. Now I am ready to move on.

My adviser says I could also try lower temperature. I inoculated the cells again, shake under room temperature for 30 hours. The cells look pretty dense now. I will prep them just to see if they will also give me the right size of plasmids. I will come to update if they do not. If I do not come to update, it means that method is successful, too. Or I am dead.

To be honest, for now I am kind of done with DH5a cells. It seems like weird things happen to me when I use them. I had an unpleasant transformation experience last year with them. I think probably my piece of DNA has some weird effect on DH5a. I wonder if it is because my dCTP deaminase is from bacterial origin so recombination happens? Or something weird is happening here. Anyway. I am done spending time on weird mini-cultures. I used to use oneshot cells and they were always great. I am thinking about moving my plasmid into oneshot cells.

Best regards,

Heng

I think I figured out why: In DH5a cells, it does not have LacI, which inhibit the Lac promoter. So Lac is always on. When I put an insert behind Lac, the promoter is always on and it makes whatever crap they read from the sequence I put in. This is why they grow in lower temperature--the toxicity is lowered in low temperature. If I put the plasmid in another strain with LacI, which is like a purse string of the lac promoter, the lac will not be always on. It will only be on when IPTG is added. So I am going to order XL1 strain which has a LacI.

See here:https://groups.google.com/forum/#!topic/bionet.molbio.methds-reagnts/DAsucuJoB-g

I am coming back with the solution of my own problem so in the future if someone else has the same question, they may waste less time to figure it out. I shook the cells at room temperature instead of 37 degrees. The cells grew slower but they did grow. I am happy with the result.