Formamide (Deionized)

For long-term storage, we recommend resuspending the RNA in stabilized formamide and storing at -70 degrees C. To remove the formamide, add 4 volumes ethanol and, if less than 20 µg RNA, also add NaCl to 0.2 M. Precipitate the RNA and use for downstream experiments.

There are many sample buffer formulations used, however we have found a distinct difference in the band appearance depending on the sample buffer composition. After evaluating urea, formamide, and various buffer systems, we found that the sharpest, flattest bands were obtained with a urea, Ficoll, and TBE buffer solution. Sample buffers made with formamide resulted in fuzzy, indistinct bands.

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Yes, formamide is supplied as a liquid even though it is sold by weight.

It is appropriate to use out of the bottle for most applications (hybridization buffer, loading buffers for sequencing gels, and formaldehyde-containing RNA gels) if less than one year old and stored properly (-20 degrees C). (If formamide smells like ammonia, it is probably breaking down and should not be used.) However, we have seen that in a protocol we have for running RNA gels without formaldehyde in the gel, freshly deionized formamide is essential for preparation of the loading buffer. That protocol as well as the protocol for deionizing the formamide follows:

These reagents are used in electrophoresis of RNA in agarose gels in 1X MOPS EDTA buffer. (Note: no formaldehyde is used in the gel.) The sample is prepared in the sample buffer (1.5 to 3 µL sample mixed with sample buffer). Heat the RNA in sample buffer for 10 min at 65 degrees C. Place on ice immediately then load gel.

10X MOPS EDTA (10X ME)

(1) Measure out 700 mL DEPC-treated water in a DEPC-treated 1-liter graduated cylinder and place in a DEPC-treated 1-liter beaker.
(2) Place beaker on stir plate and begin stirring with a DEPC-treated stir bar.
(3) Add 104.65 g MOPS to beaker. Weigh out MOPS using a sterile-flamed spatula from an unopened bottle MOPS or one set aside for RNA only. Dissolve by stirring.
(4) Add 40 mL 0.25 M EDTA to solution. Note: the EDTA solution should be made with DEPC-treated processed water.
(5) Adjust pH to 7.0 by adding 10 N NaOH (~20 mL)
(6) Bring up to 1 liter volume with DEPC-treated water.
(7) Filter solution using a 0.2 µm Nalgene filter unit.
(8) Store at 4 degrees C for six months in a DEPC-treated glass bottle. (This prevents solution from yellowing.) NOTE: The presence of yellow color does not affect the performance of the buffer.

Formamide Deionization protocol:
1. Add 1 g mixed bed, ion exchange resin for every 10 ml of formamide. Suitable ion exchange resins include BioRad AG501-X8 and Fisher Resin 1-300.
2. Stir for 30 to 60 min @ RT.
3. Filter through Whatman No. 1 filter paper.
4. Dispense into units of use and store at -20°C