Ligation:blunt vs single base overhang - an old question? (Mar/09/2007 )

People always says "single base overhang fragments are more difficult to ligate than blunt end fragments", so why go to more work to make the A overhang for TA cloning?
I did`n find any reference.
Many companies have TA cloning Kit even using T4 ligase(Topo isomerase for invitrogen).
Thanks for your attention!

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I think it is a matter of preference. I have done blunt ended cloning a few times and have
been successful in doing so. You might have to screen through a lot more colonies to check
for the insert but its doable

J

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depends what you want it for, really. Commercial TA kits are really easy to use, and they don't self-ligate which is a big problem with blunt-end cloning. Ligating a PCR product into a TA vector should be extremely simple, where have you heard that it is difficult...?

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Thank you for your reply! "Selfligation is a big problem for blunt end ligation."I think u r right.
It is, for example,
NEW ENGLAND Biolabs
The Catalog & Technical Reference:
When the cleavage of the retriction enzyme produces a single base overhange,
the book always say that.(single base overhang fragments are more difficult to ligate than blunt end fragments). Really strange！

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I thought TA cloning is way easier compared to blunt end isnt it? At least the TA cloning has special feature to clone the fragment. Blunt end doesnt have any complementary sequence. Oh well...

My understanding is sticky end ligation is the easiest followed by TA cloning and finally will be the blunt end. =)

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I think that the commercial TA or blunt-end cloning kits are made for the cloning of PCR products. If the PCR products have A overhang then we use TA cloning; if we use proofreading DNA polymerase, then we get blunt end product and we use blunt-end cloning kit.

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