Liz

Hi, So Im trying to grow my protein in Codon + RP cells in order to use them in a pull down assay. However, theyre being very tempremental and Im struggling to get all my variants to grow consistantly. They will all grow and express my proteins fine sometimes but then not a week later, despite using the same antibiotic stocks and transformation plates. This week Im having trouble with two of them. The first just wont grow although two other constructs transformed at the same time have grown fine. The second grew fine last week but wont grow this week. It was suggested that it could be a bacteriophage infection but all my other cultures all grew fine and Ive been told that you only see the effects of a bateriophage infection when you try to scale up from a mini starter culture to your main culture. These constructs dont even get that far. The starter cultures have failed 3 times now. Im going to do fresh transformations but I wondered if anyone had any ideas as to what Im doing wrong? Regularly, 1 out of my 6 transformed lines wont grow, seemingly for no reason, and with no consistancy to with construct (so I dont think any of my constructs are specifically toxic to the bacteria). I dont want to keep going in circles but this is causing me big problems in the grander scheme of the experiment Im trying to complete. Any help would be greatly appreciated! Thanks, Liz