Cloning Problem (cells grow but no plasmid isolated) - (Mar/17/2010 )

I've checked the media and grew a tube for 3 days and still had no growth. I know the miniprep kit is fine because I ran a positive control (one of my other plasmids on the same backbone) and got similar yields to previous preps. The plates and the Amp are both brand new (under a week old).

I should check the OD's and try adding glucose but the cells I'm using lack the DE3 so they shouldn't be having any leaky expression but I realize anytime there is a start codon you can get a small amount of expression but I will setup the glucose trial next week.

Many of the cloning cell lines do not have DE3, you get that in your expression host from the bacterial DNA.

http://www.csun.edu/~hcbio027/biotechnolog...c4a/petsys.html

But I have had problems in the past with cloning strains losing plasmids during culture on LB plates or liquid media in the absence of glucose. Give it a try, if it works it's a quick and easy fix.
Here's a great paper with some excellent references on plasmid stability experiments.

Low temperature and glucose enhanced T7 RNA polymerase-based plasmid stability for increasing expression of glucagon-like peptide-2 in Escherichia coli
Protein Expression and Purification
Volume 29, Issue 1, May 2003, Pages 132-139

Check out Studier's paper with recipes and nice explanations

Protein production by auto-induction in high-density shaking cultures
Protein Expression and Purification, Volume 41, Issue 1, May 2005, Pages 207-234
F. William Studier

Check your PM Your controls sound good, I'm stumped but will think on it. Good luck

Stuck with Ligation on Mar 25 2010, 10:37 AM said:

I have tried glucose stimulation now and have had no luck. The colonies that do grow have to be recombined or have the insert remove completely because I cannot find any part of my insert by PCR. This is so frustrating. I might have to start again because I have an IRES and I think the piece of DNA that is being expressed in low levels and it is toxic. I may have to redesign my insert and use a linker.

Sounds very frustrating. Have you tried checking for your insert with restriction digest? I have had enough PCR screening fail only to find the insert is present by restriction digest, to not always trust the PCR screen. Sometimes, not often, but sometimes pcr screening doesn't work.

But, it does sound like you've got other issues and will need to do some further investigation. Bummer. When having these problems, it's a good practice to run controls every step of the way, just in case there's a problem with your methods or techniques, you can rule that out and be confident that it's not you, it's the construct and or cell line that is not cooperating!! Good luck