deP for cloning - Dephosphorilation (Nov/28/2006 )

hi

I have a fragment (250bp) (Xba1/Xba1) that I want to clone in Xba1 site of a plasmid.

should I dephosphorilate one of them?


does one of them have to be not dePhos.

thanks

jm

You need to dephosphorilate your vector, so that you decrease the probability it circularizes again. You shouldn't dephosphorilate your insert because you need phosphate groups to ligate...

ok thanks

in the same perspective.
since I wante to clone a 200 fragment, I decide to order this fragment.
the max size that I can order is 100bp. Then I order the 250bp in 2 parts.

meaning 4 aligos:

as:
-------------------- ------------------------
------------------------ ---------------------

to ligate those 2 fragments Do I need to phosphorilate them?

thanks

jm

and you can buy phosphorylated oligonucleotide