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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.


Dephosphorylation of Insert

Dephosphorylation of Insert - Molecular Cloning Forum

Dephosphorylation of Insert - 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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Old 07-15-2010, 08:38 PM
Pipette Filler
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Default Dephosphorylation of Insert



Hi,
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?
Thanks
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Old 08-05-2010, 02:12 AM
Pipette Filler
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Default Re: Dephosphorylation of Insert

Hi Hannah,

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!
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