Hej,
Recently I am trying to trasnsform e.coli with pBluescript on LB(AMP)-dishes. I have run the experiment now three times - allways with the same result: E.coli grows on all plates well, except the no Plasmid control (e.coli without pBluescript). The only problem is, it also grows on the negative control.
The plasmid for the negative control is double restricted with EcoRI and BamHI and has no Insert. I tried to ligate the plasmid it before transforming it into E.coli with T4 Ligase o/n at 16°. Is there a change that those sites can ligate? Or does anyone have another clue?
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pito
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Posted 13 April 2012 - 01:12 AM
pBluescript contains an amp resistance gene.. so if it ligates, then yes, you will have resistance..
If you cut the DNA and then ad T4, then of course it will ligate again.
ANd do you check your restiction right? You check whether it is cut or not?
But a general note: I do not understand your negative control. Your negative control is a bacterium with digested plasmid?
If you cut the DNA and then ad T4, then of course it will ligate again.
ANd do you check your restiction right? You check whether it is cut or not?
But a general note: I do not understand your negative control. Your negative control is a bacterium with digested plasmid?
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.
