Invitrogen™ High DNA Mass Ladder

Catalog No.	Quantity
10-496-016	26 μg

Catalog No. 10-496-016 Supplier Invitrogen™ Supplier No. 10496016

Description

Includes

High DNAmass Ladder is supplied at 520 ng/4μL in 10mM Tris-HCl (pH 7.5), 1mM EDTA Volume: 200μL; For 50 applications

Suitable for estimating the mass (quantity) of unknown DNA samples by ethidium bromide staining

High DNA Mass Ladder consists of an equimolar mixture of six blunt-ended DNA fragments of 10, 6, 4, 3, 2, and 1Kb. Electrophoresis of 4μL of the High DNA Mass Ladder results in bands containing 200, 120, 80, 60, 40, and 20ng (520ng total) of DNA, respectively. Compatible with agarose gels. Includes tartrazine, xylene cyanol.

• Less waste, sustainable packaging

Acrylamide Gel Electrophoresis, Agarose Gel Electrophoresis, DNA and RNA Purification and Analysis, Nucleic Acid Gel Electrophoresis and Blotting

Order Info

Shipping Condition: Room Temperature

Specifications

• Content And Storage: • 200 µL High DNA Mass Ladder
  • 1 mL10X BlueJuice Gel Loading Buffer
  
  Store at -20°C.
  
  
• Concentration: 0.13 μg/μL
• Gel Type: Agarose
• Ready to Load: No
• Sample Loading Volume: 1 mL
• Size Range: 1000 to 10000 bp
• Volume (Metric): 200 μL
• Gel Compatibility: Agarose gel
• No. of Reactions: 50 Applications
• Quantity: 26 μg
• Shipping Condition: Approved for shipment at Room Temperature or on Dry Ice
• Technology: Individual chromatography-purified DNA fragments
• Product Type: High DNA Mass Ladder

Frequently Asked Questions (FAQs)

Can I know the sequences of Invitrogen DNA ladders?

Sequences of Invitrogen DNA and RNA ladders are proprietary.

Are Invitrogen DNA ladders composed of linear or circular/supercoiled DNA?

Invitrogen DNA ladders contain linear dsDNA fragments.

Are Invitrogen DNA ladders composed of single-stranded or double-stranded DNA fragments?

Invitrogen DNA ladders are composed of double-stranded DNA fragments only.

Why are the DNA bands from my molecular weight ladder smearing?

Here are a few reasons why you might see smearing of the bands:

- The DNA was degraded. Avoid nuclease contamination of DNA standards.
- Too much DNA was loaded on the gel. Decrease the amount of DNA in the gel.
- The DNA was contaminated by protein. Remove proteins by phenol extraction before electrophoresis.
- For small DNA, the bands may have diffused during staining. Add the ethidium bromide before electrophoresis.
- For radiolabeled DNA, labeling was performed by nick translation. Label the DNA by replacement synthesis with T4 DNA polymerase or label the 5' end with T4 polynucleotide kinase.
- Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30°C during electrophoresis. Check that the electrophoresis buffer used has sufficient buffering capacity.
- The DNA contained too much salt. Remove excess salt by ethanol precipitation before electrophoresis.

I'm seeing anomalous migration of my DNA ladder. What happened?

This can happen if the marker was heated. Please ensure that the ladders are not heated before use.

I need to quantitate my DNA. What ladder do you suggest I use?

Our Low DNA Mass Ladder (Cat. No. 10068013) and High DNA Mass Ladder (Cat. No. 10496016) were specifically created for quantitative estimation of DNA mass in gels.

What is the difference between the TrackIt DNA Ladders and other DNA ladders?

TrackIt DNA Ladders contain two tracking dyes in the sample buffers, which serve as visual markers for tracking electrophoresis progress through the gel, and also to indicate when maximum resolution is achieved. The tracking dyes should not obscure your visualization of DNA bands in the ladder, as the dyes run outside the limits of most DNA bands in the ladder.

TrackIt Cyan/Orange Loading Buffer is formulated with unique tracking dyes, Xylene Cyanol FF and Orange G. We recommend TrackIt Cyan/Orange Loading Buffer for DNA fragments between 10 bp and 1 kb.

The TrackIt Cyan/Yellow Loading Buffer is formulated with unique tracking dyes, Xylene Cyanol FF and Tartrazine. We recommend TrackIt Cyan/Yellow Loading Buffer for DNA fragments between 100 bp and 10 kb. The molecular weights are Xylene Cyanol FF, 638.6; Orange G, 452.4; Tartrazine, 534.4.

Note: The TrackIt DNA Ladders are not recommended for use with polyacrylamide gels and are not designed for quantitation.

I am using a quantitative DNA standard and see little difference between the bands when they are ethidium bromide stained. How can this occur and what can I do?

Here are a few suggestions:

(1) Excess volume loaded on gel. The smallest loading volume will result in the greatest difference in band thickness between different masses. Basically overloading the ladders gives saturated bands which do not show any variation in intensity. It is best to load all samples in the same volume.

(2) Sample loaded in larger or smaller volume than standard. Load equal volumes of sample and standard DNA for accurate comparison.

(3) Ethidium bromide (EtBr) staining after electrophoresis. Include ethidium bromide in the gel rather than staining after electrophoresis to increase the difference in band intensity.

(4) EtBr was in the gel, but not the running buffer. This results in the absence of EtBr in the lower half of the gel after electrophoresis and therefore differential fluorescence between bands in the upper half of the gel and the lower half of the gel.

What can cause anomalous migration of my DNA markers?

In general there are very few reasons why a DNA marker would run anomalously. Here are a few suggestions:

(1) For Lambda DNA/Hind III Fragments, the cos site reannealed. Heat Lambda DNA/Hind III Fragments at 65 degrees C for no longer than 10 min before electrophoresis.

(2) Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature <30 degrees C during electrophoresis. Check that the electrophoresis buffer used has sufficient buffering capacity.

(3) DNA was denatured. Do not heat standards (except Lambda-derived markers) prior to electrophoresis. Dilute markers in a buffer containing 20 mM NaCl.

I ran my DNA molecular weight ladder, and several bands seem to be missing. Can you offer suggestions on how to rectify this?

There are a few possible reasons for this:

(1) Small DNA bands were electrophoresed off the gel. Electrophorese the gel for less time, at lower voltage, or use a higher percentage gel.

(2) The small bands migrated with the dye front due to differences in ionic strength between gel and buffer. Be sure the buffer in the gel is the same as the electrophoresis buffer.

(3) DNA bands of similar molecular size were not resolved. Increase the electrophoresis time and check the proper percentage gel for resolution. For DNA fragments <1,000 bp, try Agarose 1000 (Cat. No. 16550100) instead of agarose.

(4) DNA was denatured. Do not heat standards (except Lambda-derived markers) prior to electrophoresis. Dilute markers in a buffer containing 20 mM NaCl.

Are there any suggestions to improve the detection of the bands of my DNA molecular weight ladder in an agarose gel?

Faint or absent DNA ladders might be due to:

(1) An insufficient quantity or concentration of DNA loaded on the gel. You can increase the amount of DNA.

(2) Degraded DNA. Avoid nuclease contamination of the DNA standards. Use sterile or autoclaved tips.

(3) DNA was electrophoresed off the gel. Electrophorese the gel for less time, at a lower voltage, or use a higher percentage gel.

(4) An improper UV light source was used for ethidium bromide - stained DNA. Use short-wavelength (254 nm) UV light for greater sensitivity.

(5) The fluorescence bleached after long UV exposure. Stain the gel again with ethidium bromide.

(6) For Biotinylated Lambda DNA/Hind III Fragments or Biotinylated PhiX174/Hinf I Fragments, the biotin was removed by exposure to alkaline conditions. Minimize exposure of biotinylated DNA to NaOH. Note: Alkaline transfers to a membrane do not present a problem when performed at room temperature.

Can I use the DNA Mass Ladders to quantitate DNA after radiolabeling?

No. The Mass Ladders comprise an equimolar mixture of six DNA fragments. Therefore all the bands would be of equal intensity after labeling.

How can I label DNA ladders?

Either T4 DNA polymerase or T4 polynucleotide kinase can be used to label the 100 bp DNA Ladder, 1 Kb Plus DNA Ladder, and the DNA Mass Ladders.

How can I quantitate DNA on an agarose gel?

For fragments between 100 bp and 2,000 bp, use the Low DNA Mass Ladder. 4 µL of the Low DNA Mass Ladder corresponds to 10, 20, 40, 80, 120, and 200 ng respectively of fragments, 100, 200, 400, 800, 1,200 and 2,000 bp.

For fragments between 1,000 bp and 10,000 bp use the High DNA Mass Ladder. 4 µL of the High DNA Mass Ladder corresponds to 20, 40, 60, 80, 120, 200 ng respectively of fragments 1,000, 2,000, 3,000, 4,000, 6,000, and 10,000 bp.

To calculate the amount of DNA in 1 µg of the Lambda/Hind III standard:
(size of band in bp/48503 bp) X 1 µg = µg of DNA in band A

For example, to calculate the amount of DNA in the 564 bp band of the Lambda/Hind III standard:
(564/48,503) X 1 µg = 0.011 µg or 11 ng of DNA in the 564 bp band.

What could be causing smearing of my molecular weight DNA ladder?

(1) Incorrect concentration of DNA ladder gives poor separation and smearing of DNA bands.
(2) Samples containing ≥50 mM NaCl, 100 mM KCl, 10 mM acetate ions, or 10 mM EDTA (i.e. certain restriction enzyme and PCR buffers) will cause loss of resolution on E-Gel agarose gels.

Safety and Handling

WARNING: Cancer - www.P65Warnings.ca.gov

For Research Use Only. Not for use in diagnostic procedures.