New England Biolabs, N3238S, Fast DNA Ladder

Related Categories DNA Markers & Ladders Applications DNA Analysis Specification Bases Fragment Mass (ng) bp 1 36 10,002 2 36 5,001 3 36 3,001 4 38 2,000 5 28 1,500 6 108 1,000 7 43 766 8 40 500 9 33 300 10 41 150 11 61 50 Effective Size Range 50bp to 10,002bp Storage Buffer 0.002% Xylene Cyanol 5 mM Tris-HCl 5 mM EDTA 5% Glycerol pH 8 @ 25°C FAQ Q: How much Fast DNA Ladder should I load on a gel? A: For a standard electrophoresis system, we recommend loading 0.5 µg (20 µl) of the Fast DNA Ladder on the agarose gel. For a fast electrophoresis system (5 to 30 minutes separation), follow the system’s manufacturer recommendations: 5 to 20 µl load. A dilution of the ladder may be required. Q: What percentage of agarose gel should I use? A: The best separation occurs on a 1.2% TAE agarose gel, but you can also use a TBE gel. Below 1%, the 50bp fragment may not separate from the 150bp fragment. Q: Can I store it at -20°C? A: For long term storage, you can store at 4°C or -20°C. If stored at -20°C, mix well after thawing, as the dye and the Ficoll-400 may form a gradient in the vial. Q: Why are there extra bands visible on polyacrylamide gels? A: To provide increased intensity for the smaller bands and the reference bands, multiple fragments of the same size have been cloned into the plasmids used for many of the DNA ladders. These fragments, identical in size, are indistinguishable on agarose gels, but, on acrylamide gels, even slight differences in DNA sequence can lead to noticeably different migration rates. Fragments of the same size do not always run the same on acrylamide. For the 100bp DNA Ladder, the 500 and 517bp fragments, which run as a close doublet on agarose, separate very clearly on acrylamide, with the 517bp band running around midway between the 500 and 600bp fragments. This “anomalous” migration is an inherent characteristic of acrylamide gel electrophoresis and does not indicate any error in the stated size of the DNA fragments in our ladder. For the 50bp DNA Ladder and the Low Molecular Weight Ladder, two or more of the bands comprising the 200bp reference band tend to run differently, resulting in one or more extra bands detectable around the 200bp range. As with the 100bp DNA Ladder, this is attributed more to the limitations of acrylamide gel technology than to a problem with the ladder composition. As defined in most molecular biology lab manuals (Maniatis’ Cold Springs Harbor Molecular Cloning Manual, 2nd edition) and as acknowledged by the manufacturers of acrylamide gels, applications requiring precise sizing of DNA fragments should be performed using agarose gels whenever possible. Q: Does the Fast DNA Ladder work with the FlashGel® DNA System or the E-Gel® Precast Agarose Electrophoresis System? A: The Fast DNA Ladder has been tested and is suitable for FlashGel® 1.2% gels, E-Gel® 0.8, 1.2 and 2% gels with ethidium bromide or SYBR dye. This DNA ladder is also suitable for the E-Gel® EX 1% and 2% gels, the E-Gel® NGS, the E-Gel® SizeSelect and the E-Gel® CloneWell gels, but may require a ½ to ¼ dilution in water for optimum results. Q: Can I use SYBR® and/or GelRed® dyes with the DNA Ladders from NEB? A: Because high affinity nucleic acid binding dyes can affect DNA migration during electrophoresis, post-staining of gels with SYBR and GelRed dyes is highly recommended. Post-electrophoresis staining results in superior sensitivity and eliminates the possibility of dye interference with DNA migration. While agarose gels can be precast with these dyes, the migration and/or resolution of some DNA ladders may be affected. Therefore, some DNA ladders may require optimization/diution when run on gels precast with these dyes. For optimum results, please follow the dye manufacturer’s recommendations and protocols carefully, specifically concerning the loading amounts. We recommend using our 1 kb Plus DNA Ladder for Safe Stains (NEB #N0559) when using SYBR or GelRed as precast dyes, as this DNA ladder was specifically optimized for this application. Additionally, avoid using loading dyes that contain SDS, as SDS might cause an abnormal band pattern in precast gels. We recommend using Gel Loading Dye, Purple (6X), no SDS (NEB #B7025) for this application.” Q: Why is my DNA Ladder floating out the wells? Why are there no bands visible in my DNA ladder gel lane? A: If the ready-to-load DNA Ladder or the 6X Loading Dye supplied with the ladder has been frozen at any time, the Ficoll will form a density gradient in the vial upon thawing. Any volume pipetted from the top of the vial will contain only a small amount of density agent and will float out of the wells. To prevent this, mix well upon receipt and before use.