Advantages of multiple restriction enzymes for cloning

AI Thread Summary
Utilizing dual restriction enzymes for cloning offers significant advantages over single enzyme use, particularly in preventing self-ligation of the vector, such as pBluescript II. By employing two enzymes, researchers can ensure the correct orientation of the DNA fragment, which is crucial for proper protein expression. Additionally, pretreating the cut vector with a phosphatase can further minimize the risk of self-ligation, enhancing the efficiency of the cloning process.
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why would utilizing duo restriction enzymes for cloning be better then just one?
( e.coli to pBluescript II )
 
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That way the vector can't self-ligate.
 
thanks a lot Monique
 
You can prevent self-ligation fairly effectively by pretreating the cut vector with a phosphatase. One major advantage of using two restriction enzymes for cloning is that you can control the orientation that your DNA fragment is inserted into the vector (especially useful if you're making a construct to express protein).
 

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