RNA Ligation

Gen Yan Yang gyang at MED.UNC.EDU
Tue Dec 29 18:46:34 EST 1998


Hi, Tim,

Thanks a lot for your information. Have you tried the higher ligation 
temperature, for example,  17 degrees for a shorter time(16 hours) using 
your ligation mixture?

I have checked every reagent in my ligation mixture except for RNA 
ligase. After incubating in 37 degrees for 2 hours, no degraded RNA can 
be observed. However, the RNA is not intact after ligation. At first, I 
doubt if I put too much RNA ligase, so this time I just use 50u (5ul) in 
my ligation mixture. Unfortunately, RNA also degraded. I treated all the 
stuffs used for RNA operation and I am very careful to avoid introducing 
RNase by myself. I am confused what is the culprit. Tonight I check the
RNA ligase by incubating RNA with it in 17 degrees for 16 hour.

Do you think in what degress DMSO or PEG can improve the ligation
efficiency ?

tfitzwater at NEXSTAR.COM wrote:
> 
> 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)

-- 
Gen-Yan  Yang
CB# 7090, 436 Taylor Hall
The University of North Carolina at Chapel Hill
Chapel Hill, NC. 27599-7090 , USA.
Phone:  (919) 966-4436 (work)
        (919) 929-4365 (home)
Email: gyang at med.unc.edu



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