{"product_id":"neb-n3031l","title":"New England Biolabs, N3031L, pBR322 DNA-BstNI Digest","description":"  \u003cb\u003eRelated Categories\u003c\/b\u003e DNA Markers \u0026amp; Ladders  \u003cb\u003eApplications\u003c\/b\u003e DNA Analysis  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eBases\u003c\/b\u003e Fragment Mass (ng) bp 1 426 1,857 2 243 1,058 3 213 929 4 88 383 5 28 121 6 3 13  \u003cb\u003eEffective Size Range\u003c\/b\u003e 13bp to 1,857bp  \u003cb\u003eStorage Buffer\u003c\/b\u003e 10 mM Tris-HCl 1 mM EDTA pH 8 @ 25°C  \u003cb\u003eFAQ\u003c\/b\u003e 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: 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. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835494944937,"sku":"N3031L","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-n3031l","provider":"Iright","version":"1.0","type":"link"}