deionised formamide - deionised formamide (Oct/25/2004 )
Is there any body who can tell me how to make "deionised formamide" for Northern blot? Or it is conmercial only?
Most aqueous formamide solutions become acidic (pH 5) with time. Since the reaction is alkaline-catalyzed, it is best to buffer at pH 7 with phosphate or PIPES. RNA shows some phosphodiester breakage on contact over several days.
Traditional deionization introduces water (50% of the wet resin is water) which causes an increase in conductivity after 2 hours exposure and which affects nucleic acid hybridization and melting temps. Adding extra resin does more harm than good. (Samanta and Engel, 1987)
1. Obtain a 500 mL bottle of formamide.
2. Weigh out 25 g (5%) Bio-Rad AG501-X D) or MB-3 resin.
3. Lyophilize resin overnight to complete dryness.
4. Combine with formamide and stir gently for 3 hours (not more than 7 hours).
5. Filter through Whatman #1 prerinsed with deionized formamide.
6. Aliquot and store at –20°C. Stable at least 1 year. Conductivity of deionized formamide is ~ 1.5 µmho using wet resin after 2 hours and ~ 0.5 µmho using dry resin after 3 hours. A270 1 cm path 100% formamide < 0.15. A 50% aqueous solution < 7.5 pH.
I have had good results using EM brand formamide without deionization.