Preparation of Denaturing Agarose Gels
A variety of denaturants can be used with agarose. Alkaline gels are most often employed with single stranded DNA because pouring and handling such gels is not only nonhazardous, but convenient as well. However, because strong alkali will hydrolyze it, formaldehyde is used with RNA.
Preparing Alkaline Agarose Gels
At high temperatures, alkaline conditions will hydrolyze the Agarose polysaccharide chains. To prepare an alkaline gel, the agarose is first melted in water and cooled to near gelling temperature. Buffer concentrate is then added and the gel is poured. GEL PREPARATION
SAMPLE PREPARATION: Mix samples with an equal volume of 60mM NaOH, 2mM EDTA, 20% Ficoll and 0.06% Bromocresol Green. The stock solution consists of 2g Ficoll, 0.4ml 50X alkaline buffer, 6mg Bromocresol Green and enough deionized water to bring to 10 mL. |
Preparing Formaldehyde Agarose Gels (for RNA Analysis)
Note: RNA is subject to rapid degradation by RNase present in the environment. For optimal results, use water treated with DEPC. GEL PREPARATION:
SAMPLE PREPARATION:
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