Invitrogen™ TrackIt™ 100 bp DNA Ladder

Here are a few suggestions:

(1) Excess volume loaded on gel. The smallest loading volume will result in the greatest difference in band thickness between different masses. Basically overloading the ladders gives saturated bands which do not show any variation in intensity. It is best to load all samples in the same volume.

(2) Sample loaded in larger or smaller volume than standard. Load equal volumes of sample and standard DNA for accurate comparison.

(3) Ethidium bromide (EtBr) staining after electrophoresis. Include ethidium bromide in the gel rather than staining after electrophoresis to increase the difference in band intensity.

(4) EtBr was in the gel, but not the running buffer. This results in the absence of EtBr in the lower half of the gel after electrophoresis and therefore differential fluorescence between bands in the upper half of the gel and the lower half of the gel.

In general there are very few reasons why a DNA marker would run anomalously. Here are a few suggestions:

(1) For Lambda DNA/Hind III Fragments, the cos site reannealed. Heat Lambda DNA/Hind III Fragments at 65 degrees C for no longer than 10 min before electrophoresis.

(2) Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30 degrees C during electrophoresis. Check that the electrophoresis buffer used has sufficient buffering capacity.

(3) DNA was denatured. Do not heat standards (except Lambda-derived markers) prior to electrophoresis. Dilute markers in a buffer containing 20 mM NaCl.

There are a few possible reasons for this:

(1) Small DNA bands were electrophoresed off the gel. Electrophorese the gel for less time, at lower voltage, or use a higher percentage gel.

(2) The small bands migrated with the dye front due to differences in ionic strength between gel and buffer. Be sure the buffer in the gel is the same as the electrophoresis buffer.

(3) DNA bands of similar molecular size were not resolved. Increase the electrophoresis time and check the proper percentage gel for resolution. For DNA fragments <1,000 bp, try Agarose 1000 (Cat. No. 16550100) instead of agarose.

(4) DNA was denatured. Do not heat standards (except Lambda-derived markers) prior to electrophoresis. Dilute markers in a buffer containing 20 mM NaCl.

Faint or absent DNA ladders might be due to:

(1) An insufficient quantity or concentration of DNA loaded on the gel. You can increase the amount of DNA.

(2) Degraded DNA. Avoid nuclease contamination of the DNA standards. Use sterile or autoclaved tips.

(3) DNA was electrophoresed off the gel. Electrophorese the gel for less time, at a lower voltage, or use a higher percentage gel.

(4) An improper UV light source was used for ethidium bromide - stained DNA. Use short-wavelength (254 nm) UV light for greater sensitivity.

(5) The fluorescence bleached after long UV exposure. Stain the gel again with ethidium bromide.

(6) For Biotinylated Lambda DNA/Hind III Fragments or Biotinylated PhiX174/Hinf I Fragments, the biotin was removed by exposure to alkaline conditions. Minimize exposure of biotinylated DNA to NaOH. Note: Alkaline transfers to a membrane do not present a problem when performed at room temperature.

(1) Incorrect concentration of DNA ladder gives poor separation and smearing of DNA bands.
(2) Samples containing ≥50 mM NaCl, 100 mM KCl, 10 mM acetate ions, or 10 mM EDTA (i.e. certain restriction enzyme and PCR buffers) will cause loss of resolution on E-Gel agarose gels.