Gen Yan Yang
gyang at MED.UNC.EDU
Tue Dec 29 18:46:34 EST 1998
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
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)
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Email: gyang at med.unc.edu
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