Need urgent advise.. - (Apr/29/2013 )

Hello,
I am doing cloning in the traditional manner using pcambia1300 destination vector but I am facing several problems with it.
1.after ligation ---> electropration --->dh5alpha cells---. overnigth at 37C---.NO COLONIES, but if i keep it there for 4-5 days I can see the colonies.
Later If i use these colonies for colony pcr i can see the product from one part of my insert. but the same colony i also use for streaking but i dont get colonies there ever, even in 5- days no colonies.
2. this time instead of PCR i thought of using colony from plate and put it in liquid culture for 2 days, I mimpreped it but get no plasmid....
a.Is it because its not the right plasmid or is it like log phase passed away and i cannot get the plasmid anymore, I dont knw the reason.
How should i proceed further with getting the right vector with insert and use it for sequencing?

Thanks in advance
Monika

First of all, can't trust the colonies that you got after 4-5 days at 37'c, bcoz after 4 days we cant expect the selection antibiotic on plate are stable, and so the possibility of contaminated colonies are more.

the positive PCRs can be the product that carried over from ligation and hence on the plate during transformation. to avoid this use primers one at the vector and other in the insert for colony PCR.

so it doenst surprise me that you got no plasmid, as i think the colonies you got are'nt containing the actual plasmids you are looking for.

so you may have to repeat the ligation with all possible controls at ligation and transformation.

thanks for your suggestion but this experiment i performed almost 4-5 times and always get the same results.But yes i will try with new primer set and then repeat ligation and transformation all over again.

Have you tried the experiment with half of the normal working concentration of your antibiotic?