PCRBIO Ladders I-IV are manufactured using a combination of plasmid restriction digest fragments and PCR amplification products. The ladders are resuspended in sample buffer ready for immediate gel loading. The buffer formulation also allows for greater room temperature stability compared to conventional DNA ladders.
Optimal ladder loading
Use of undiluted ladders (5–10 µL) is recommended for clear and consistent results with all dyes and agarose concentrations, except when using GelRed Stain and GelRed Loading Buffer for which loading 5-10 μL of 1:10-diluted ladder will yield best and most accurate results.
Increasing the amount of ladder loaded on a gel causes a shift in the migration of ladder bands of the same molecular weight. This is more pronounced with some dyes (e.g., GelRed Loading Buffer) than others. Thus, care should be taken when comparing apparent molecular weight of DNA targets estimated in comparison to different amounts of DNA ladder.
Optimal agarose concentration
Ladder I and II: Are best used on gels with ≤1.5% agarose concentration. Running these ladders above this concentration will prevent separation of higher molecular weight bands and is not recommended.
Ladder III and IV: Resolve well on ≥1.5% agarose gels.
Ladder III does not resolve well below 500 bp on 1.5% agarose gels, except when 10 μL of diluted ladder is used with GelRed dyes or 10 μL of undiluted ladder is used with the Midori Green Advance stain. This ladder is ideal for high-resolution applications and should only be used when detailed band separation below 1500 bp is required and ideally run on 2-2.5% agarose gels. Longer running times are required for optimal separation of lower molecular weight fragments.
Ladder IV showed well-separated bands across all conditions but performed best with 2% agarose and GelRed stain. This ladder also resolves well on 1.5% and is recommended for most applications requiring basic size estimation of low molecular weight (<1500 bp) bands. It is a good alternative to Ladder III if extremely precise fragment size estimation isn’t required.
Optimal gel staining dyes
GelRed Prestain Loading Buffer provided the best balance of signal and resolution across all gel concentrations and amounts of ladder loaded, although the DNA concentration-dependent run may cast some doubts on the real size of the fragment of interest.
GelRed Nucleic Acid Stain worked well when diluted ladder were used and enabled good band resolution and uniform intensity across the different gel concentrations. As such, it is probably the best option for use with PCRBIO DNA Markers and is also cost-effective because only small volumes of dye are required per gel.
SYBR, EcoDye and Midori Green dyes all give good results when undiluted ladders are used, but care should be taken to run the various ladders on the recommended agarose concentrations (see points above). Also important to note, the intensity of the bands will decrease with longer runs more than with the GelRed dyes.
Other standard gel staining dyes may also be used, including ethidium bromide. We recommend following the manufacturers instructions with these and testing PCRBIO Ladders by running 5 and 10 μL of both undiluted and 1:10 diluted (in the supplied 6x Sample Loading Buffer A) ladder to identify optimal loading conditions with any dye not described above.
