Isolation of Total RNA from just about anything

Chris Pittock pittock at rsbs0.anu.edu.au
Mon Jul 11 20:12:29 EST 1994


acc4636 at tamsun.tamu.edu (Anthony C.) wrote:

[lots deleted]
>I am using the following 
> recipe :
> 
> 11 ul of RNA (10 ug!!)
> 9 ul of formaldehyde
> 5 ul 10X MOPS
> 25 ul formamide
> 5 ul of premade gel loading buffer from SIGMA
> 
> The gel is
> 
> .5 grams of wide range agarose from SIGMA
> 36 ml of depc treated double distilled water
> 5 ml of 10X MOPS
> 9 ml of formaldehyde
> 5 ul of 500 ug/ml EtBr
> 
> The gels are a little too soft (pouring buffer on sometimes rips the
> wells) so Im going to halve the formaldehyde today.  The 5 ul of EtBr
> is a little too bright so Im going to use 2 ul.  The MOPS is a 10X powder
> from sigma that you just add 1 liter of water to (minimize the things that
> can go wrong :) ).

A couple of years back I ran too many RNA denaturing gels. I made them as
follows:				                      Yours were:

1.5% agarose							               1.0%
5% formaldehyde	                  18% (?!)
1x Mops buffer																				1x
33 ug etBr in 50ml gel            2.5 ug/50ml  (?!)

I was loading 20ug total RNA per lane - As you can see, the formaldehyde in
your gels is very high in comparison (doesn't this make them v. brittle?),
you try to visualise half as much RNA with less than a thirteenth of the
amount of ethidium bromide.  Unless there is a reason for using small
amounts, I would scale that up considerably.

Sample treatment:

-> heated to 65 degC 15min,                      -> someone mentioned
-> chilled on ice,										                     90-100 deg to 
-> stop solution  (pretty standard,              denature RNA - 
50% glycerol, 1mM EDTA pH8, 0.25% w/v            really necessary???
Bromophenol blue & same for xylene
cyanol FF).

I trust that this is of some help (or at least a data-point to see what
poeple do to their RNA).  All the best.

																	               Chris.



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