RNA ligation

Gen Yan Yang gyang at MED.UNC.EDU
Sun Dec 27 12:22:52 EST 1998


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)

Any suggestion will be greatly appreciated.



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