formamide loading buffer - storage? (Apr/09/2010 )
I've read some storage conditions of formamide loading buffer: 95% formamide, 20mM EDTA, .05% BPB---most say to store at 4C and long term storage at -20C.
My 100% formamide stock is kept at RT, as the bottle instructs. Why do I need to keep the loading buffer at 4C? Why would something like this go bad?
If you are doing northern blots, you want to use deionised formamide. This is stable for about 6 months at -20 deg C as a frozen solid before the formamide becomes ionised again, and causes the frozen stock to melt (apparently). You can deionise formamide over an ion exchange column, but it is much easier to buy it in fresh unless you are used to using columns.
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, J Biochem Biophys Methods. Deionization of formamide with Biorad AG501-X(D). 1987 Aug;14(5):261-6.)
1. Obtain 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. 50% aqueous solution < 7.5 pH.
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.
Having supplied the protocol, I must add that I don't bother with deionization anymore. Formamide breaks down to formic acid, which does not freeze at -20°C. I store my formamide at 4°C. Stick your formamide in the freezeer overnight. Pour off any liquid in the morning. Thaw and use what's left.
Formamide loading buffers contain water and should be kept at -20°C where they are good for several years. Discard when the tube is no longer completely frozen at -20°C.