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Transformation help - (Mar/06/2006 )

Hi guys!
I have done the blue-white assay and I got blue colonies on my plasmid plate but no colonies on my insert+plasmid plates, what does that mean, that the ligation or digestion might be wrong?


i guess you purify your plasmid from a gel, so you get cut plasmid only anyway. do you excise your insert from another plasmid (blunt end or sticky?) or do you PCR it? if you excise it blunt-end, it can be that the plasmid re-ligates and the insert doesn't go in. try sticky end ligation if possible or try SAP (Shrimp alkaline phosphatase) on the plasmid before you ligate it.
and have you checked if your ligase works? try self-ligating your cut plasmid. if you don't get colonies, then your ligase is crap (expired maybe?).
hope that helps!


QUOTE (mads @ Mar 8 2006, 12:26 PM)
i guess you purify your plasmid from a gel, so you get cut plasmid only anyway

This depends on the distance between the restriction enzymes you use... If they are relatively close you can not distinguish between double cut or single cut plasmid.

So, please tell us you complete strategy for cloning...


let me know one thing your plasmid got blue colonies means is it digested and ligated plasmid or without doing this transformed directly, in this case if second statement is correct your ligation is not perfect means may be your ligation buffer lose the ATP by multiple thawing or your ligase is efficiency is decreased or it may seazed because of presence of some chemical compounds
for this after digesting the plasmid your treating with cip enzyme if yes your insert lose the p groups because of presence of phosphatases in your sample
u r not satisfy with above answers give complete details what u did ok