New England Biolabs, N3233S, Low Molecular Weight DNA Ladder

Related Categories DNA Markers & Ladders Applications DNA Analysis Specification Bases Fragment Mass (ng) bp 1 42 766 2 27 500 3 20 350 4 33 300 5 27 250 6 110 200 7 33 150 8 43 100 9 58 75 10 63 50 11 43 25 Effective Size Range 25bp to 766bp Storage Buffer 10 mM Tris-HCl 1 mM EDTA pH 8 @ 25°C FAQ 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: Why is the separation of the lower bands incomplete? A: The low molecular weight ladders contain small fragments and require long run times to resolve the bands effectively. To visualize the smaller bands, a higher percentage gel is helpful (up to 3% agarose, which is most effectively performed using a specially formulated low melt agarose). Smaller bands also tend to diffuse more than larger bands as the electrophoresis is being carried out, so running faster (at higher voltage) can be useful, as long as not too much heat is generated. In most systems, gels of 2% or higher can be run at a voltage of 200 for several hours without excessive heat damage. Q: What are the overhangs on the DNA ladder fragments? Can I end-label them using the T4 polynucleotide kinase (PNK)? What about the Klenow fragment? A: All fragments in the 1 kb, 100bp, 1 kb Plus, 50 bp, Low Molecular Weight DNA Ladders and the PCR Marker have 5’ overhangs that can be end-labeled with the T4 PNK (NEB# M0201) or filled in using the Klenow Fragment (NEB# M0212). Labeling with PNK can be done by the standard protocol of removing the existing phosphate groups from the fragments ends using CIP or the Antarctic phosphatase, but we also had excellent results using the much simpler “exchange” reaction, which utilizes the ability of the kinase to remove existing phosphates from the DNA and replace them with labeled phosphates. The procedure that worked best for us is as follows: -1 μl T4 PNK -1 μl 32P ATP (3,000Ci/mmol, 5mCi/ml) -2 μl 10x T4 PNK buffer -1 or 2μl DNA ladder (1μg) Add distilled water to 20 μl. Reaction is carried out at 37°C for 30 minutes. Run the samples for 50 to 60 minutes at 100V in TBE buffer in a 4-20% acrylamide gel (10cm x 10cm). A 20 minutes exposure gives very readable signals. The signal strength is about twice that signal when ADP is added to 100 μM. Q: Why are the DNA ladders showing up on my Southern blot? What is the sequence or composition of the ladder bands? A: This phenomenon has been observed in a variety of labs, and, while we do not have an absolute explanation, there are 2 likely sources for the seemingly unlikely hybridization of the user’s probe to the ladder’s DNA. One is that, if the probe itself was generated from a pUC or pBR based plasmid, there may be a low level of plasmid contamination in the probe that is hybridizing to the ladder’s bands, most of which have pUC-like sequences in them.Alternately, there may just be some random, low specificity homology between the probe and some segment of the ladder fragments. The bands consist entirely of DNA derived from either pUC or Adenovirus 2 DNA, which is used as filler DNA to generate plasmids of the right size for the markers. Because the molar amount of DNA in the ladder bands is frequently in great excess compared to the DNA being probed, even a relatively low homology can still result in enough incorporation of probe to give a readily detectable signal. 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: 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: Can I use GelRed® with the Low Molecular Weight and 50 bp DNA Ladders from NEB? A: Yes. Using GelRed for post-electrophoresis staining is highly recommended for optimal performance. Staining for 30 minutes is usually sufficient for proper visualization of the DNA fragments. While agarose gels can be pre-cast with GelRed, the migration and resolution of DNA ladders might be affected. According to the manufacturer, high affinity nucleic acid binding dyes can alter the DNA migration during electrophoresis. For the Low Molecular Weight and 50 bp DNA Ladders specifically, which are made up of sticky-ended DNA fragments, the ladder bands will likely smear when gels are pre-cast with GelRed. Post-staining with GelRed results in superior sensitivity and eliminates the possibility of dye interference. These ladders, however, will run as expected with pre-cast gels if they first undergo a blunting treatment (through a polymerase fill-in or nuclease blunting reaction).