Poly(A)+ selection

kim at M44.UNM.EDU kim at M44.UNM.EDU
Mon Dec 9 16:48:57 EST 1991


Hi Group:

  I seem to have a few questions regarding poly(A)+ selections, so I thought I
would try to pull them together here.  

  So far, it seems that the "type"numbers for oligo(dT) cellulose refer to the
length of the (dT) tails.  This may have some bearing on the binding capacity
of the cellulose, or it may not.

  In my hands, I cannot get good recoveries off of columns run using SDS in the
buffer, nor can I make batch methods work.  Since this is a simple
binding/elution reaction, I would think that the column geometry is not as
critical to the outcome as it is in gel-filtration columns in which molecules
of different size must be separated.  If this is so, then would an Eppendorf
tube with a small hole in the bottom work as well as a small column as a 1 ml
syringe?

  I have been trying to figure out how much total RNA can be loaded onto
a given amount of cellulose.  I looked in Maniatis, which says that a 1-ml
packed volume (0.2 to 0.5 grams, I think)  should be sufficient for 10 mg of
total RNA.  In Methods in Enzymology, they calculate the capacity from the
published capacity of 100 A(260) units per gram.  With some assumptions
factored in, a figure of 10 mg of _messenger_RNA is given!  Over two orders of
magnitude greater than the estimate given in Maniatis.  In a batchwise 
protocol given by Promega, the loading capacity of oligo(dT) cellulose is given
as 500 micrograms of total RNA for 0.3 g of cellulose.  Stratagene, however,
takes 1 gram of cellulose, suspends it in 20 ml of DEPC-water, and uses 920
microliters in a column made from a 1 ml syringe.  This is loaded with up to
0.5 mg total RNA.
  
  The capacities given by Promega and Stratagene seem to be the closest match,
more or less (0.3 g per 0.5 mg RNA vs 0.1 g per 0.5 mg RNA).  These figures
still make up a large ballpark.  Is experimentation the only way to figure how
much RNA to load on a given column?

  Washing steps are sometimes done by gravity and somethimes by centrifugation. 
Volumes of wash range from 1 x column volume to 10 x column volumes.  Is a
small wash with centrifugation as good as a larger wash by gravity?  I'd rather
do a small, fast wash for the sake of time.

Thanks for all the help so far, and in advance for help on this.

Daniel Kim   KIM at FLOVAX.LANL.GOV





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