RNA Ligation

tfitzwater at NEXSTAR.COM tfitzwater at NEXSTAR.COM
Tue Dec 29 17:21:48 EST 1998


I routinely use 80-100 Units of BMB T4 RNA ligase per 40 ul reaction in the
 presence of 10% DMSO at 4 degrees C. for 18-72 hours.
Have not tried PEG in the reaction.

Tim Fitzwater

------------
From: gyang at MED.UNC.EDU (Gen Yan Yang)
Subject: RNA ligation
Date: 27 Dec 1998 09:22:52 -0800

Hi all,
Does anyone have experience to use T4 RNA ligase to ligate RNA
substrate? I want to know what's the approprate amount of T4 RNA ligase
in the reaction mixture. It's reported 2u/ul, so 50ul reaction volume
will add 100u ligase. I use the following reaction, just use 50u ligase:

5.0ul 10x RNA ligase buffer
1.0ul RNA Oligo (4.6ug)
5.0ul T4 RNA ligase (10u/ul, from BMB)
1.3ul RNAsin (40u/ul, from BMB)
25.0ul 40% PEG
12.7ul mRNA (4ug)





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