T4 RNA ligase - Question about substrate (Jul/24/2009 )
I would like to ligate a ssDNA oligonucleotidic adaptor to a ssDNA fragment in order to perform a PCR with a primer complementary to the adaptor sequence and a primer designed on the ssDNA fragment. I've read that the T4 RNA ligase can ligate ssDNA fragments as well as RNA fragments.
Has anybody ever tried performing a ligation of this kind? I would like to try using the Fermentas T4 RNA ligase (#EL0021) (simply because I've a trial stock...), does anybody know if it is a good enzyme? But, primarily, I need the protocol!!!!!
Should work fine, i used T4 Ligase on dsDNA but i heard you can use it on RNA as well. I haven't used the brand you are going to try, but ive been using Biorads T4 DNA Ligase
T4 RNA ligase is a different enzyme than T4 DNA ligase. The RNA ligase should be able to ligate oligos to ssDNA. The 5' end of the fragment needs to be phosphorylated for ligation to occur (that is the end of the fragment participating in the ligation site).
The NEB one that i have states:
Catalyzes the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini in duplex DNA or RNA. This enzyme will join blunt end and cohesive end termini as well as repair single stranded nicks in duplex DNA, RNA or DNA/RNA hybrids (1).
although ive only used i with dsDNA
Is it able to create RNA/DNA hybrids?
I have used several times the T4 RNA ligase from fermentas (#EL0021) to ligate RNA adapters to RNA and it worked very well.
andrea massimo on Jul 31 2009, 01:35 PM said:
Have you ever tried using it to ligate DNA oligos to RNA molecules?
Yes, it was a modified (5' adenylated) DNA adapter which is used to make small RNA libraries. I ligated it to a small RNA pool without ATP in the reaction. I also used the RNA ligase to ligate a chimeric DNA/RNA adapter to the product of previous ligation.