Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

PIR 1 Competent Cells - Poor Transformation Efficiency (Feb/09/2010 )

Has anyone had problems with tranformation efficiency when purchasing these cells? There is a single supplier and I have had nothing but problems with these cells. I am about to make my own cells, but do not have time to do that on a regular basis. When I call the company supplying the cells, they claim that they do not have problems with efficiency. They are not a commonly used strain and the replacement lot is the same as the bad lot they sent in the first place. They do not know when they will have a new lot available. Yes, I have transformed using pUC19 to test efficienty and it is horrible- with no colonies at all the last time I tested. I would just like to validate with someone else that I have spent megabucks for useless cells, and wasted a lot of time. GT115 has its own set of problems. Unfortunately, with the R6K origin, these cells are the only option. I have been using these cells for the last 10+ yrs- it is not a matter of technique etc. I have tested a ligation mix that previously gave a ton of colonies with a previous lot of cells and got 1 colony. This is very frustrating.


I accidently found the key to Pir1 cells in frustration. We optimized the zeocin dose and got no colonies. I was so frustrated I didn't even bother to throw them away. I just shoved them back into the incubator and took two days off and then the weekend came. That Monday when I came in to throw out the plates, I had big fat colonies! I ran a mini after I streaked a new plate for each colony picked and got no DNA. I was again frustrated. But the next day, I ran another mini on the newly streaked plate( which the colonies grew over night), and got DNA! So from now on I leave the plates for two day instead of one. You can see teenie tiny colonies on day one, but they are very robust on day two. This goes against all cloning procedures but it works.

-Candace Johnson-