No colonies- toxic or unligated?

Mélanie Tremblay mel_4_7 at yahoo.ca
Tue Jan 27 20:46:37 EST 2004


Hi

I amplified my gene (2.3kb) with primers containing restriction site 
(and 6 extra bases), after I digested with NcoI and XmaI and ligated in 
vector (digested and dephosphorylated). Few colonies (about 10) were 
produced in XL-10 and no one positive with control positive ok.
After that, I tried TOPO TA cloning. I followed protocol and obtained 
few colonies (around 10) with no one containing insert (with TOPO10F').

My insert is probably toxic, but how I can know if it's the cloning that 
failed or it's the bacteria that not support it? This gene is presently 
encoded in pbluescript in XL-10 cells. Growth is slow but I can produce 
a little bit of DNA.


I'm desperated!!! Can you help me?
Mélanie Tremblay




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