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topo cloning


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#1 sharifah

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Posted 28 October 2004 - 04:20 AM

hi,
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......... :lol:

#2 rassen

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Posted 28 October 2004 - 11:53 AM

OK, you have white colonies on antibiotics plates, which means that you did get positive plasmid with insert. When you inoculate them in LB medium, they failed to grow. Now the problem lies at the medium or the conditions you grew your bacteria. Is your medium freshed prepared? Did you shake the container virgorously at 37C?

#3 sharifah

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Posted 14 December 2004 - 01:45 AM

i tried again. i have lots of white colonies again but for 2 times, i did not get any plasmid from the clones i selected. I was very sure my preparation was correct as i did a control togehter with that by preapring puc19 miniprep and i got a lot of palsmid from that. this happened to me twice. i am still trying to do topo cloning but it was either lots of whiote colonies but no plasmid in it or very few white colonies and not growing in lb tet (5). I incubated all of the cultures at 37 for sure........
any suggestion will be most welcome, thanks.




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