Measuring Molecular Weight with SDS-PAGE
The mobility (Rf) of a molecule in gel electrophoresis is determined by its free solution mobility, Y0 (= mobility in a gel of zero %) and the sieving action of the gel matrix. In denaturing protein electrophoresis, the addition of SDS to the electrophoresis buffer uniformly coats the proteins with negative charges, equalizing the charge…
Read MoreCasting Gradient Gels
Gradient gels are cast with a higher concentration of acrylamide at the bottom than the top. Gradient gel applications include the determination of protein molecular weights and the separation of molecules which co-migrate on uniform gels. Casting of gradient gels requires a gradient forming apparatus, and is more labor intensive than casting uniform percentage gels.…
Read MoreGel Preparation for SDS-PAGE
Two categories of buffer systems are available for SDS PAGE: continuous and discontinuous. Continuous systems use the same buffer in both the gel and tank. While continuous gels are easy to prepare and give adequate resolution for some applications, bands tend to be broader and resolution consequently poorer in these gels. Discontinuous buffer systems employ…
Read MoreSample Preparation for SDS-PAGE
SDS is a powerful detergent, which will solubilize many cells and tissues. This greatly facilitates sample preparation for SDS PAGE because most samples will be completely dissolved by heating to 95°C in loading buffer (detailed below). A somewhat stronger loading buffer, containing more SDS and dithiothreitol (DTT) at a higher pH, can be used for…
Read MoreGel Electrophoresis of RNA & Post Electrophoretic Analysis
Agarose electrophoresis of RNA requires the inclusion of denaturing agents in the gel. In the absence of denaturants, RNA assumes compact secondary structures, which distort the relationship between molecular weight and mobility.Urea, used as a denaturant in polyacrylamide gels, disrupts the hydrogen bonds which hold the agarose gel together, and alkaline conditions, used in denaturing…
Read MoreRNA 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 MorePFGE 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 MoreIn 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 MoreDNA/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 MoreRestriction 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,…
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