pBLuescript transformation - (Aug/19/2008 )
Hi everyone,
I'm having problem with cloning in pBluescript. I've narrowed down the problem, and I think the problem is in transformation step. In transformation, in addition to the recombinant pBluescript, I also include empty pBluescript and nontransformed cells. I used LB plates + Ampicillin 100 microgram/ml for selection as well as blue-white selection. What I had was overgrown plates, and all cells were white in all plates (controls as well). I change the Ampicillin with freshly prepared stock, and this time I got nothing, even in cells transformed with empty pBluescript 
. I used the same concentration of Ampicillin as before. Should I lower the concentration? Thanks in advcance for any suggestion
DNAbug
-DNAbug-
QUOTE (DNAbug @ Aug 19 2008, 07:31 PM)
Hi everyone,
I'm having problem with cloning in pBluescript. I've narrowed down the problem, and I think the problem is in transformation step. In transformation, in addition to the recombinant pBluescript, I also include empty pBluescript and nontransformed cells. I used LB plates + Ampicillin 100 microgram/ml for selection as well as blue-white selection. What I had was overgrown plates, and all cells were white in all plates (controls as well). I change the Ampicillin with freshly prepared stock, and this time I got nothing, even in cells transformed with empty pBluescript . I used the same concentration of Ampicillin as before. Should I lower the concentration? Thanks in advcance for any suggestion
DNAbug
I'm having problem with cloning in pBluescript. I've narrowed down the problem, and I think the problem is in transformation step. In transformation, in addition to the recombinant pBluescript, I also include empty pBluescript and nontransformed cells. I used LB plates + Ampicillin 100 microgram/ml for selection as well as blue-white selection. What I had was overgrown plates, and all cells were white in all plates (controls as well). I change the Ampicillin with freshly prepared stock, and this time I got nothing, even in cells transformed with empty pBluescript
. I used the same concentration of Ampicillin as before. Should I lower the concentration? Thanks in advcance for any suggestionDNAbug
Hi,
Probebly you may need to decrease the concentration. I sugest to check different concentrations and see which one is better. I have the same problem with sigma freshly prepared amp and have decided to do that.
-saraarasus-