New England Biolabs, N3012L, Lambda DNA HindIII Digest

Related Categories DNA Markers & Ladders Applications DNA Analysis Specification Bases Fragment Mass (ng) bp 1 477 23,130 2 194 9,416 3 135 6,557 4 90 4,361 5 48 2,322 6 42 2,027 7 12 564 8 3 125 Effective Size Range 125bp to 23,130bp Storage Buffer 10 mM Tris-HCl 1 mM EDTA pH 8 @ 25°C FAQ 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: How do I use the lambda DNA-Hind III digest? A: We recommend loading 1ug (2ul) of the Lambda DNA-Hind III. The fragments can be separated effectively on agarose gels ranging from 0.6 to 1% using TBE or TAE buffers. To prepare the samples, put 2ul of the marker into 13ul TE buffer (10mM Tris-HCl, 1mM EDTA) and add 3ul of 6X loading buffer for a final volume of 18ul. Heat the samples to 60C for 3 minutes and remove to ice or load immediately on the gel. Loading more than 1ug will result in the larger bands appearing very broad and therefore more difficult to use for accurate sizing. Loading less than 0.5ug makes the smaller bands harder to visualize. Q: Why does my Lambda DNA molecular weight marker appear smeared and not represent the correct banding pattern? A: These banding aberrations are almost always a result of the preparation of the DNA molecular weight marker for electrophoresis. For example, the Lambda DNA markers (Hind III and BstE II digests) require a brief heating before electrophoresis (we recommend 3 minutes at 60C, followed by placing on ice or loading directly onto a gel) to dissociate the 12-base sticky ends of the cohesive ends. Without this heating, the 23kb band and the 4.3kb band of Lambda-Hind III anneal by H-bonding between the complementary bases to form a 27kb band. Visually, this results in a decrease in intensity of the 4.3kb band. Without heating lambda DNA-BstE II, the 8.4kb band and the 5.6kb band anneal to form a 14kb band. Visually, this results in a decrease in intensity of the 8.4 and the 5.6kb bands plus the appearance of a 14kb band.One caveat to heating DNA markers is that double stranded DNA requires a minimal amount of ionic strength to keep it from denaturing. This denaturing of strands may result in irregular or fuzzy banding patterns. Therefore, we do not recommend diluting DNA markers in water, but rather in a buffer with minimal ionic strength such as TE buffer. 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.