G-50 Columns
2.5 grams of G-50 into 50 ml of T.E. Use a blue cap tube.
--Autoclave for 15-20 minutes
--let cool.
--keep refrigerated
TO USE:
(1) put a small hole in a the bottom and top of a microtube (1.5mls)
--use a syringe needle heated in gas flame to make hole
(2) add glass beads or glass wool to bottom ( apprx. 100-150 ul)
(3) fill tube will G-50
(4) Place micro tube into 7 ml tube (this tube will collect the desired fractions).
(5) pre-spin* the tube for approx. 1-1.5 min so G-50 fills approx. 75% of microtube and is not a solution but instead a "dry" paste ( looks like a free-standing column with slanted top)
(6) load up to 100 ul of solution onto center of "dry"G-50, careful so that the pipette tip does not disturb your easily crumbled free-standing column
(7) put microfuge tube into a new 7ml tube
(8) spin* for 2.5 -- 3 mins. Large molecules are spun through G-50, unicorporated nucleotides remain in column.
(9) discard column as dry radioactive waste (if you used any radioactivity...)
*spin in a clinical centrifuge at highest setting
HYPERLINK "http://genomics.rsmas.miami,edu" http://genomics.rsmas.miami.edu Marine Genomics Laboratory
updated 2001 Douglas L. Crawford
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