random alkaline RNA ladder and RNaseT1 digestion - need help

Rimas Rimantas.Plaipa at gf.vu.lt
Mon Mar 12 06:52:45 EST 2001


Dirk Müller wrote:
> 
> for analyses of my ribozymes i need to carry out an RNaseT1 digestion and an
> alkaline digestion of the RNA to build an RNA ladder. I found protocols in
> the literature but as it seems to be normal with published protocols - they
> dont't work.

And so will be with protocols which I will give to you so I will discuss
possible culprits first.

There are two different T1 activity units used by commercial providers.
Some sell it at >300000 U/mg, some 50000 U/mg, but the real difference
is the unit used, not the specific activity of enzyme. And anyway you
need to try various concentrations and find out the best.

Alkali solutions get "rotten" when stored for long time, especially
diluted ones in small volumes (by absorbing CO2 from air in overcrowded
laboratories).

Urea solutions should not be subjected to >40 C during preparation . Be
careful if you try to speed up the dissolution or thawing and of course
don't autoclave them.

My optimised protocols:

T1:

Get RNA dry in eppendorf tube (dry some solution in vacuum, precipitate
with EtOH etc)
+4 ul solution of  urea with buffer  ( 700 ul of 10M urea with XC and
BPB and 1 mM EDTA+100 ul 200 mM Na citrate pH5.0)
shake to disolve RNA and spin down
+1 ul of T1 solution in 50 % glycerol with some pH5.0 buffer
5 min at 55 C.

Alkaline:

get RNA dry ?
+ 4 ul ( 600 ul 10M urea with dyes and 1mM EDTA + 200 ul H2O)
shake?
+ 1 ul 200 mM NaOH
80 C 0.5-2 min 

Conc of T1 and incubation times need to be adjusted individually.

Urea solutions may be stored indefinitely at room temp , T1 - at -20 C.
NaOH have to be replaced when appropriate (extent of  cleavage declines
or have nothing else to do)
---------------
Rimantas Plaipa,

Department of Biochemistry and Biophysics,
Vilnius University,
Ciurlionio 21/27, Vilnius 2009, Lithuania.

E-mail: rp010gf at voruta.vu.lt
Phone: (370-2)-650381 
Fax: (370-2)-235049


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