Formamide-dye mix

To terminate the reaction the capillary contents are ejected into 5yu.l formamide dye mix (99% deionized formamide containing 10 mM EDTA, 0.2 mM NaOH, 0.1% xylene cyanol FF dye). After denaturation at 100°C for 30 s the samples are applied directly into gel wells and electrophoresis carried out by the usual method (Section 3.1.2.). Seif et al. (1980) give a recommended order of loading ... 

Expel the contents of each capillary tube into 15 pi of dye-formamide-EDTA mix in four Eppendorf tubes. Heat in water bath 90-100°C for 3 min. 

Add 200 pi of cold 70% EtOH, seal again. Brief spin, aspirate dry. Air dry. Add 3 pi formamide loading dye. Seal with regular tape, mix thoroughly on multitube vortexer, spin down. 

All experiments were started from 5-pg testis total RNA and 5-pg lung total RNA per sample. This was first amplified by T7 in vitro transcription, and 5-pg aRNA was used for every labeling reaction. After labeling with Cy3/Cy5 dye, both samples were mixed, and for every experiment, identical amounts of sample, i.e., 5-, 20- or 40-pmol Cy3/Cy5 dye, were applied. The cDNA was further dissolved in a solution containing 50 % formamide, 25 % buffer, 15 % Mouse COT, and 7.5 % polydT35. This cDNA mixmre was then respectively denamred at 96 for 3 min, set on ice for 2 min, heated to 42 for 5 min, centrifuged at m = 12,000 rpm for another 5 min, and subsequently held on ice before applying it on the microarray slide. 

Since the lower phenols are volatile, they are best separated after conversion into involatile, coloured derivatives (azo dyes) (see p. 664) [37, 87, 154]. The TLC of the azo compounds derived from other simple alkyl phenols also has been described in these articles [37, 87]. Eaesel-guhr layers, impregnated with formamide, have been used as well as strongly alkaline silica gel layers. After separation, the coloured derivatives can be quantitatively eluted from the layer and determined [154]. Mixtures of alcohols and phenols can be satisfactorily chromatographed as their dinitrobenzoates [38]. Numerous phenolcarboxylic acids have been separated on mixed layers of silica gel G and cellulose, using the solvents toluene-ethyl formate-formic acid (50 + 40 + 10) and chloroform-acetic acid-water (80 + 20 + 20). Phenolcarboxylic acids differing little in polarity, can be better separated if the layers are saturated with moisture before use [158]. 

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