MIDI's low recovery or vector issues? - (Jun/02/2008 )
Hello, Im´from Argentina, so I'll try to be clear since my english is a little limited.
When I do the transformation and plate the transformants, I obtain a great number of colonies after the ON incubation, but when I transfer a colony to LB for 16-24 hs, the growth is lesser than expect. When I do a MINI prep, I obtain as much as 0.08 mg/ml; with a MINI prep I obtain 0.1-0.2 mg/ml or less!!
I´m working with the p3XFLAG-CMV from Sigma, with an insert of 1 kb (ERK2). This construction have previously used in my lab without any problems. In fact, I made the transformants from a one year old MIDI obtained in this lab under identical conditions. I test two different strains with my construction (XL-1 an DH5-a) and I have the same results. I've transformed these strains with another plasmid and I have great growth (so the strains are OK) and the MINI and MIDI extractions are the expected (0.1 an 1 mg/ml respectively, so the kits are also OK). The ampicilin is ok, and so the LB.
Anyone have an opinion about these?? I don't know what to do, or where is the problem!
Although I can not explain why, I've had similar problems with specific constructs just not amplifying well. No matter what, I just can't seem to get a high yield with either a mini or midi. As mentioned, this problem only occurs with specific constructs. I use more culture than the instructions say. I spin down media until I get a good size bacterial pellet. This may help a bit but I always wind up having to EtOH precipitate the DNA from multiple midi preps. This way I can resuspend the precipitated DNA in a small volume of water to get a concentrated stock of plasmid.
Thank you! I'll try to do that and see what happens.