dsDNA clean-up with Sephadex

Michael Benedik bchs1b at ELROY.UH.EDU
Wed Nov 20 14:01:39 EST 1991


In article <1991Nov19.184320.391 at vaxa.strath.ac.uk>, cbar89 at vaxa.strath.ac.uk writes:
>Does anyone out there have a suitable method for the removal of nucleotides
>and, if possible, 20 mer primers from a reaction mixture using Sephadex
>in a home made spin column. The dsDNA in the mixture is at least 120 bp and 
>requires some concentration as well as clean up.
>The sample should also be desalted in the clean up step.
>
>Any help as to column preparation, spin times and g-force would be greatly
>appreciated.
>
>
>Martin J Pearce
>Forensic Science Unit
>University of Strathclyde
>Glasgow UK.
>
>PS Any suggestions for alternative techniques are welcome. (But we haven't
>got a centrifuge that will take Centricon devices!!)

I make homemade spin columns in a 1 ml syrenge with a little glass wool
stuffed in the bottom (can use silicon wool if you want to be sure it is
not sticky) and fill syringe full of column matrix. Can use sephadex G-50
we prefer biorad P-10. Put the column in a 15 ml falcon tube, spin at 2000 RPB
oops, 2000 RPM for 2 minutes, the matrix should now look dry. cut the lid off
a 1.5 ml microtube and put in bottom of falcon tube, put syringe end inside
1.5ml tube, load sample, and spin 2000RPM for 2 minutes. I have found this
really only  works reliably in a swinging bucket rotor. Speed and time are
variable, but do both spins the same. Discard syringe and your sample is in
the micro tube. This matrix works well for desalting and removing
nucleotides. If you want to remove linkers can use other matrices, some
commercial spin columns use CL2b, CL4B and CL6B to separate larger oligos
from each other. I have no experience with those, but they should work. Just
need to use a strong bead like an acrylamide bead so that it does not crush
like agarose beads do.


	---------------------------------------------------
	 Michael Benedik
	 Department of Biochemical and Biophysical Sciences
	 University of Houston
	 INTERNET: Benedik at UH.EDU	BITNET: Benedik at UHOU



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