Posted 28 October 2004 - 04:20 AM
i have just use a TopO TA cloning kit to clone my tet(M) resistant cassette. I PCR the tet DNA using Promega Taq Polymerase and then purify using QIAquick column. Then follow the invitrogen protocol for topo TA cloning and electroporate into Top10.
I have a lot of blue and white colonies even in 50 ul plate. But when I selected 10 colonies and grew them i LB + 5 ul tet, NONE of them grow.........
Is it because i did not gel extract my pcr product?? Or is it because there iwas self ligation of the vector? but how can it be because i selected only white colonies????
I just do not know whyyyyyy?? Please help.........
Posted 28 October 2004 - 11:53 AM
Posted 14 December 2004 - 01:45 AM
any suggestion will be most welcome, thanks.