Please help - Why can these can/cannot be ligated?

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Please help - Why can these can/cannot be ligated?

Post by techbrit » Tue Mar 15, 2011 12:24 pm

1. Which of the following pairs could be ligated together? (All ter- mini are cohesive and complementary.)
a. 5'-dephosphorylated linear insert DNA + linear vector
b. Supercoiled vector + linear insert DNA
c. 5'-dephosphorylated linear vector + linear
5'-dephosphorylated insert DNA
d. Linear 5'-dephosphorylated vector + linear insert DNA
e. Nicked vector + linear insert DNA

In general, which of the above possibilities would be the best approach in a subcloning experiment like the one you have done? Why?

I am struggling understanding why ligation is /is not possible. Thank you so much in advance.

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Post by JackBean » Tue Mar 15, 2011 2:05 pm

if at least one is phosphorylated, then it's fine, because the ligase will ligate only one strand and the other will be handled by the bacteria after transformation.
Both, the insert and vector, must be linear, otherwise the ligase won't be able to work, of course. I'm not sure about the nicked vector, whether is it nicked on one strand or both strand e.g. 4 nucleotides far apart or 20 nt apart or what?
Usually, the vector is dephosphorylated. If it wasn't, it could be self-ligated (i.e. ligated empty; or e.g. two vectors ligated together) and then you got circular vector, which has resistance, but not your gene of interest. Dephosphorylating the insert doesn't make that much sense, because even if it was self-ligated, it won't have the resistance, so you won't get false-positive colonies ;)

Cis or trans? That's what matters.

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