T4 RNA ligase - Question about substrate (Jul/24/2009 )
andrea massimo on Aug 5 2009, 11:41 AM said:
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?
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
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.
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:
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!!!!!