New England Biolabs, N0558S, TriDye™ Ultra Low Range DNA Ladder

Related Categories DNA Markers & Ladders Applications DNA Analysis Specification Bases Fragment Mass (ng) bp 1 49 700 2 46 650 3 28 400 4 63 300 5 28 200 6 11 150 7 14 100 8 21 75 9 70 50 10 10 35 11 16 25 12 19 15 13 125 10 Effective Size Range 10bp to 700bp Storage Buffer 10 mM Tris-HCl 10 mM EDTA 10% Glycerol 0.006% Xylene Cyanol 0.006% bromophenol blue 0.06% Orange G pH 8 @ 25°C FAQ Q: How much of the TriDye™ Ultra Low Range DNA Ladder should I load? A: We recommend loading 5 ˜µl (0.5 µg) of TriDye™ Ultra Low Range DNA Ladder per gel lane on polyacrylamide gels. We recommend loading 10 µl (1 µg) of TriDye™ Ultra Low Range DNA Ladder per gel lane on agarose gels. Q: Why do I only see 12 bands in the Ultra Low DNA Ladder? A: On agarose gels, the 700 bp and 650 bp DNA fragments might run as a single band. They will run separately on polyacrylamide gels. Q: Should I use TBE or TAE buffer for my agarose gels? A: We recommend using TBE buffer in the agarose gels and electrophoresis buffers, as TBE buffer allows for a better resolution of the smaller DNA fragments. Fragments below 15 bp might not separate efficiently when using TAE buffer. Q: Can I use the TriDye™ Ultra Low Range DNA Ladder with GelRed® or SYBR® Dyes? A: We recommend using GelRed®, GelGreen®, SYBR® Safe, SYBR® Gold and SYBR® Green I as post-electrophoresis stains only. Using them as dyes precast in the gels might interfere with the migration of the smallest DNA fragments and result in an abnormal band pattern. Optimization might be required for proper visualization of all DNA fragments. Intensity of some bands might vary. Q: Which polyacrylamide gels can I use? A: The TriDye™ Ultra Low Range DNA Ladder can be fully separated on native 4-20% TBE, 10% TBE and 20% TBE polyacrylamide gels. This ladder is not suitable for use in denaturing conditions. Q: Why are some of the bands in the DNA Ladder fainter at the bottom of the gel? A: Most stains run in the opposite direction from that of the migrating DNA. As a result, the bottom of the gel might have a lower concentration of stain, which might result in un-uniform staining of the bottom bands. How much of the gel will end up with a lower stain concentration is highly dependent on the migration conditions (gel percentage, voltage, buffer, run time, etc.). This effect can be counteracted by adding some stain to the running buffer. When using the DNA ladder for agarose gel electrophoresis, we recommend using ethidium bromide at 0.5 ug/ml in both the gel and the migration buffer, for proper visualization of the smallest DNA fragments. We do not recommend casting gels exceeding 5mm in thickness, as the smaller DNA fragments might diffuse more easily and might be harder to stain efficiently in thicker gels. If using wide combs (10 mm), we recommend loading 15 µl (1.5 µg) of TriDye™ Ultra Low Range DNA Ladder per gel lane for efficient visualization of the smallest DNA fragments. For optimal visualization of all the bands, we recommend exposing the gel to UV without any casting tray whenever possible. Even UV-transparent trays can obscure DNA fragments under UV exposure. Q: Why is the 10bp fragment fainter than the other fragments? A: Even though the DNA mass per band is higher for the 10 bp fragment than the other fragments, it will appear fainter on the gel, due to the fact that very small DNA fragments do not bind intercalating agents as well as others, that they diffuse a lot more throughout the gel matrix, and that the charged intercalating agents (ethidium bromide or others) will run in opposite direction in the gel.