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| DNA Techniques Post questions and discuss DNA techniques and protocols such as DNA extraction, PCR, and the study of DNA-binding proteins. |
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#1
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| When attempting to create a recombinant DNA molecule, by inserting a foreign sequence into the vector plasmid; what is the point (and how does it achieve it) of treating the plasmid DNA with alkaline phosphatase? I think it has something to do with the orientation of the inserted sequence, but unsure how the treatment achieves this. |
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#2
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| Here is my understanding of phosphatases: Vectors are normally treated with alkaline phosphatases, such as CIP, following a digestion in order to prevent re-ligation of the vector in the absence of the insert. The phosphatase removes the 5' phosphate group so that it cannot interact with the 3' hydroxyl group. Thus, when a ligation is performed you increase your likelihood that your vector contains your insert and not just an empty vector. I hope it helps! |
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#3
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| phosphatase is used to remove phosphate group from the 5'end to prevent self ligation. |
| Tags |
| alkaline , dna , making , phosphatase , plasmid , recombinant , treat |
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| Thread | Thread Starter | Forum | Replies | Last Post |
| plasmid with secreted alkaline phosphatase | Gaël Yvert | Yeast Forum | 0 | 06-10-2008 11:40 AM |