Considerations for Loading Pulsed-Field Gels (PFG) with High Molecular Weight DNA

When samples are prepared for loading on pulsed-field gels, they are mixed with loading dye and water, and are thus diluted. As described in "Homogenization of High Molecular Weight DNA (HMW DNA) Samples,” samples require time to disperse and relax following dilution. Failure to allow samples to fully relax before loading onto the gel can result in smearing and trailing of the DNA. As described, samples should be mixed by pipetting with a wide bore pipette tip and incubated at 37°C for 30 minutes, or several hours to overnight at room temperature, after diluting with loading dye. Ensure that loading dye does not contain SDS, as SDS can cause the samples to float out of the well by introducing air bubbles into the sample. It is also recommended to use wide bore pipette tips with hydrophobic coating, especially when working with UHMW DNA (DNA that was prepared using low agitation speeds). These coated tips facilitate loading in the PFG wells by preventing the DNA from sticking to the pipette tip, which could otherwise lead to unintentionally pulling the DNA sample out of the well. 




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