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|Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here.|
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Dephosphorylation of Insert
By mistake I added Shrimp Alkaline phosphatase to my insert digestion and not to my plasmid digestion.
My plasmid is ~7kb and insert is ~550bp, and they are digested with 2 different restriction enzymes HindIII and NdeI.
Should I go ahead and ligate and transform or is it not worth it?
Re: Dephosphorylation of Insert
The dephosphorylation reaction is done with the plasmid to prevent vector from re-sealing to itself during the ligation. If you added it to your insert by mistake, you might still be ok because the plasmid (vector) should still be phosphorylated. It's worth a try. Sorry if this is too late but hope it helps for future reference!
|dephosphorylation , insert|
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