Archive for August 2011

Native DNA PAGE gels can be used to detect small mutational differences between DNA molecules. In heteroduplex analysis, for double stranded DNA, the basis of separation is the conformational difference arising from the bending of the rodlike double helix caused by small mismatches between the strands. In SSCP analysis, single stranded DNA molecules are fractionated…

Methods of DNA/RNA Purification from PAGE Gels. Polyacrylamide gel electrophoresis yields individual bands of extremely high purity, in that only one nucleic acid sequence tends to be present in each. Electrophoresis can thus serve as a powerful purification tool for DNA or RNA. In practice, most purifications are carried out on DNA, which is more…

Protein bound to a small piece of DNA will alter the electrophoretic mobility of that DNA fragment. This allows the analysis of protein-DNA interactions, including the measurement of binding rates, affinity, and specificity. In addition, bound and unbound DNA may be isolated from the gel and used for further types of analysis such as methylation…

Gel electrophoresis of PCR products is the standard method for analyzing reaction quality and yield. PCR products can range up to 10kb in length, but the majority of amplifications are at 1kb and below, where PAGE analysis is the most effective. In the size range from 400 to 1000 bases, the choice of native PAGE…

PCR amplification follows an exponential curve until a saturation point is reached, after which further amplification often serves only to degrade purity. Curve shape depends upon the amount of substrate present initially. PCR reactions produce product in a nonlinear pattern (See figure below). Amplification follows a typical exponential curve until some saturation point is reached.…

The polymerase chain reaction (PCR) is a powerful technique which uses repetitive cycles of primer annealing, primer extension, and product denaturing to produce an exponential increase in the copy number of the target DNA. Two primers are used, which flank the region of interest (See figure below). In the presence of a thermostable polymerase, the…

An exploded view of a gel cassette. Native PAGE gels are prepared by mixing an acrylamide/bisacrylamide monomer concentrate (AccuGel 19:1 or 29:1), buffer concentrate, and water to achieve the desired gel concentration. TEMED and ammonium persulfate are added to initiate polymerization and the solution is poured into a cassette. The comb is then inserted. The…

Sample preparation for native PAGE is straightforward. Since the DNA does not need to be denatured, one is concerned mainly with buffer content, density, and visibility. The salt content of the sample should be adjusted to be no greater than the 200mM salt concentration in the running buffer. Gross imbalances in salt content between sample…

In the absence of denaturants double stranded DNA retains its double helical structure, which gives it a rodlike form as it migrates through a gel (for non-denaturing electrophoresis of single stranded DNA, see SSCP Analysis). Double stranded DNA of up to 1000 bp can be separated on polyacrylamide gels. DNA over 1kb is generally fractionated…

Uracil or methylation interference assay. End labeled probe is modified at one site per molecule, and allowed to bind protein. Bound and unbound populations are separated, and strands are cleaved at the modified bases. Bases critical for protein binding will not appear as bands in the bound population. Both the methylation and uracil interference assay…