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#1 anonymous



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Posted 02 October 2001 - 09:00 PM

I think that depends on a couple of things. There are different kits with apparently different enzyme mixes for example. The topo kit from invitrogen recommends 5 minute ligations and the novagen blunt ended cloning kit recommends 15 minutes at 22C. A couple hours using standard methods should be enough though. Another thing to consider is efficiency. If you just need to clone a single fragment and find a positive clone (like it sounds like you are doing) then a short time is all you need because you only need to find one clone by your screening procecdures. On the other hand, if you were trying to construct a plasmid library, you would want to let the ligation run longer to get the highest effeciency possible since you would want to get a good representation of as many different clones as possible. Hope this explanation helps.

#2 peter



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Posted 02 October 2001 - 09:00 PM

For ligating a 800 bp insert directionally into MCS of a commercial vector, is it really necessary to allow reaction to proceed overnight, or are 2-3 hours enough? Using the shorter time obviously saves an entire day. Are the longer reaction times really only needed for blunt end cloning?

Thank you

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