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Preligation of 2 fragments

Preligation of 2 fragments - Molecular Cloning Forum

Preligation of 2 fragments - 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 01-07-2014, 11:26 PM
Pipette Filler
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Default Preligation of 2 fragments



Hi everyone

I was wondering if it's a good idea to preligate 2 fragments together before cloning them into the vector?
What I wanted to do was to preligate the fragments in 10Ál of reaction mix, heat inactivate the T4 DNA ligase and add this to a 20Ál mixture for ligation into the vector.

Could this work?

Thanks!
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Old 02-16-2014, 10:24 AM
Pipette Filler
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Default Re: Preligation of 2 fragments

It can work, but why would you inactivate ligase before the next ligation step? Just add the vector and a bit of fresh ligase.
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