DNase I Footprinting

The process of DNase I footprinting is outlined below: DNase I Footprinting Preparing the DNA substrate: DNA to be analyzed must be cloned in such a way as to present a restriction site for an enzyme leaving a 5′ overhang (3′ recessed OH) 50-150 bp from the putative binding site. This site is labeled by “filling…

Read More

RNA Electrophoresis

Electrophoretic analysis of RNA presents unique challenges. RNA is isolated in single-stranded form, without complementary sequences. It must be fully denatured in order to obtain fractionation based on size. However, RNA molecules form complex and in some cases very stable secondary structures, which are more difficult to denature than DNA. Additionally, RNA is extremely vulnerable…

Read More

PFGE and FIGE

Standard agarose gels can resolve DNA fragments up to 75 kb. Often, analysis of genomic DNA requires resolution of megabase (mb) fragments. Fragments greater in size than one megabase all run at the same rate on agarose gels, (“limiting mobility”), and are therefore not resolved from each other. This reflects the overall mechanism of sieving…

Read More

In Gel Enzyme Reactions

In many cases, the processing of DNA by enzymes is not impeded by agarose. Such reactions can be run directly in bands excised from low melting point agarose gels. The excised band is melted, mixed with the required buffer and enzyme, and then incubated at the optimal reaction temperature. The gel may solidify during the…

Read More

DNA/RNA Purification from Agarose Gels – Electroelution

The most popular alternative to glass powder elution for the complete purification of DNA from agarose is electroelution. Because agarose gels are run in a horizontal apparatus, the gel can be manipulated during a pause in the run. This allows variations of electroelution to be performed that are not possible with vertical gels, which are…

Read More

Restriction Digest Mapping

Restriction mapping involves the treatment of a DNA fragment with restriction enzymes both singly and in combination. The electrophoresis of cleavage products yields a map of the DNA in terms of restriction sites. Restriction endonucleases are enzymes that cleave double-stranded DNA at specific sites, generally 4, 6 or 8 base palindromic sequences. Because, in action,…

Read More

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…

Read More

Preparation of Agarose Gels

Use the tables below corresponding to the AquaPor agarose of choice to determine the percentage gel to cast. (LE = general-purpose; LM = low melting; ES = extra strength; HR = high resolution). AquaPor LE (all-around performance) Gel % Size Range (bp) 2 200-3,000 1.75 250-4,000 1.5 300-5,000 1 400-12,000 0.75 1,000-23,000 AquaPor LM (low…

Read More

Agarose Gel Electrophoresis of DNA and RNA – Uses and Variations

Agarose electrophoresis is used for a variety of purposes. Specialty grades of agarose have been developed to fulfill specific requirements. The most commonly used variant is low-melting agarose, which has been modified to lower its melting temperature from over 90°C to around 65°C. This allows bands to be excised from a gel and then melted…

Read More

Agarose Gel Electrophoresis of DNA and RNA – An Introduction

DNA and RNA strands are extremely large macromolecules. A 1 kilobase piece of single-stranded DNA or RNA has a molecular weight of 330,000 daltons, larger than the vast majority of proteins. Often in the molecular biology laboratory, genomic DNA fragments even as large as 1000 kilobases (1 megabase) must be separated by gel electrophoresis. The…

Read More