13 replies to this topic

[marek82313]

Hello everybody,

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!!!!!

[dreww]

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

[phage434]

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).

[dreww]

my bad

The NEB one that i have states:
Description:
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

[marek82313]

Is it able to create RNA/DNA hybrids?

[phage434]

Yes.

[marek82313]

Wonderful!!!  

[andrea massimo]

I have used several times the T4 RNA ligase from fermentas (#EL0021) to ligate RNA adapters to RNA and it worked very well.

[marek82313]

andrea massimo, on Jul 31 2009, 01:35 PM, said:

I have used several times the T4 RNA ligase from fermentas (#EL0021) to ligate RNA adapters to RNA and it worked very well.

Have you ever tried using it to ligate DNA oligos to RNA molecules?

[andrea massimo]

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.

[marek82313]

andrea massimo, on Aug 5 2009, 11:41 AM, said:

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.

Could you please give me the protocol?

I've read that is suggested the addition of 40% PEG, is it so important for the reaction?

[andrea massimo]

The protocols for ligating the adenylated and the DNA/RNA adapters are from Bartel lab, and you can find them on this site (follow the path: protocol, RNA, microRNA, miRNA cloning) or on this link
http://web.wi.mit.ed...gUpdate0705.pdf
Note that both ligations are quite different from the one you have to do. You may find more useful the protocol for the second one which include ATP in the reaction.

[marek82313]

Thank you!  

[texsequencer]

Hi Marek,

When doing a ligation with an adenylated linker you want to make sure to use T4 RNA Ligase 2. Its a truncated version that is specific for adenylated linkers / adaptors. I've used cat# 512105 T4 RNA Ligase 2 from BiooScientific and the adenylated adaptor cat #510201 from the same company and they work well.

This is the protocol that I use each time:

50-200 units of T4 RNA Ligase 2
900 ng adenylated adaptor
12% PEG 8000 MW
1X T4 RNA Ligase Buffer
200 ng ssRNA
---------------------
for a total of 20ul
incubate 2 hours at room temperature. This should give you 70-100% ligation.

Best of luck



  

marek82313, on Jul 24 2009, 04:19 AM, said:

Hello everybody,

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!!!!!

Edited by texsequencer, 30 October 2009 - 02:54 PM.