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#1
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Posted 25 October 2004 - 12:36 PM
Is there any body who can tell me how to make "deionised formamide" for Northern blot? Or it is conmercial only?
Thanks.
Thanks.
#2
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Posted 26 October 2004 - 03:41 PM
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.
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.
Edited by tfitzwater, 26 October 2004 - 03:41 PM.
