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Recreate original plasmid by cutting out insert


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#1 schaapje_86

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Posted 23 January 2013 - 06:03 AM

Maybe this is a stupid question, but I am going to ask it anyway:

I would like to cut out an insert from my plasmid (pET28c) between restriction sites Nhe1 and Not1 and ligate the plasmid back to it's orginial state without the insert (no I don't have the original plasmid anymore). However, obviously these restriction sites don't generate compatible sticky ends. Does anyone know how to recreate this plasmid?

Many thanks!

#2 pito

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Posted 23 January 2013 - 06:51 AM

Maybe this is a stupid question, but I am going to ask it anyway:

I would like to cut out an insert from my plasmid (pET28c) between restriction sites Nhe1 and Not1 and ligate the plasmid back to it's orginial state without the insert (no I don't have the original plasmid anymore). However, obviously these restriction sites don't generate compatible sticky ends. Does anyone know how to recreate this plasmid?

Many thanks!

A hint:
Klenow polymerase
Making blunt ends.
Do a google search, you should be able to find helpfull information.
If something is not clear, just ask, but I think if you look those two things up, you will know what to do!

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 phage434

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Posted 23 January 2013 - 06:53 AM

One question is why you want to do this. Usually, a plasmid with an insert is just as useful as the original (perhaps easier to use, since the insert is typically larger).

To do it, I would anneal two oligos which formed the matching cut ends and the intervening sequence, phosphorylate them (either with PNK or by adding 5' phos to the oligo order), and ligate to the cut plasmid.




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