New England Biolabs, N3019S, λ DNA-Mono Cut Mix

Related Categories DNA Markers & Ladders Applications DNA Analysis Specification Bases Fragment Mass (ng) bp 1 100 48502 2 79 38416 3 69 33498 4 62 29946 5 51 24508 6 49 23994 7 35 17053 8 31 15004 9 21 10086 10 3 1503 Effective Size Range 1,503bp to 48,502bp Storage Buffer 10 mM Tris-HCl 1 mM EDTA pH 8 @ 25°C FAQ Q: How can I quantify the amount of DNA in each band of a marker? A: The amount of DNA in each band can be determined by dividing the size (bp) of the band in question by the size (bp) of the uncut DNA molecule and multiplying this number by the total ug of DNA marker loaded on the gel. Q: General information on Lambda DNA Mono Cut Mix: A: The Lambda DNA Mono Cut Mix requires either Pulsed Field Gel Electrophoresis or, for regular gel electrophoresis, a very low percentage TAE agarose gel to effectively separate the fragments. Some common mistakes are running the gel too fast, using the wrong percentage of agarose, using the wrong buffer or loading too much of the marker.For the PFGE on a CHEF system, we recommend using a 1% agarose gel in 0.5x TBE buffer, which must be run with a cooling system, with pulse times ramped from 0.5 to 1.5 seconds, for 20 hours, 15C, 6V/cm (about 200V). For the conventional gel electrophoresis, use the following conditions: 0.5ug Mono Cut DNA, 0.4% agarose gel in 1x TAE buffer, 10V, room temperature for 24 hours. Basically, the gel must be run very slowly to achieve separation of the larger fragments. Adding Ethidium Bromide to the gel and the running buffer at a final concentration of 0.5ug/ml will effectively stain the bands during electrophoresis. Q: Can I use Midori Green with the DNA Ladders from NEB? A: NEB DNA Ladders, including Quick-Load Purple 1 kb Plus DNA Ladder (NEB #N0550), are compatible with Midori Green dye with no interference observed. Please follow dye manufacturer's recommendations exactly. Due to low dye intensity, you may need to load between 1 and 2 ug of the DNA ladder to achieve good visualization under standard UV illumination conditions. 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. Q: Why am I seeing poor results when running the PFG marker on my pulse field gel? A: There are several factors that lead to poor results for PFG markers. Here is a list of recommendations for ensuring a sharp high quality banding pattern: Use Chef DR-II system with a hexagon grid 120 degrees (do not use field inversion, 180 degrees). Use fresh 0.5X TBE for the running buffer and the gel. Use high quality water (Milli Q) for making the buffer and the gel. Load the marker as solid plug (never melt the plug). Load a thin slice of the marker, about 1 gradation, on the syringe (a thick plug will overload the well with DNA and lead to smeared blurry pattern). Always run the recommended program for each specific marker. The programs are optimized for resolving bands in the size range corresponding to each marker. Interchanging programs with different markers will often result in a poor resolution. Store the marker at -20°C.