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Buffers and Reagents

6X Orange DNA Loading Dye

Product information

Electrophoresis of tracking dyes in
6X Orange DNA Loading Dye

Features

• Two-color tracking of DNA migration during electrophoresis.
• No DNA masking during gel exposure to UV light.
• EDTA binds divalent metal ions and inhibits metal dependent nucleases.

Applications

• Analysis of small DNA molecules.
• Preparation of DNA ladders, markers and samples for loading on agarose or polyacrylamide gels.

Description

6X Orange Loading Dye is used to prepare DNA markers and samples for loading on agarose or polyacrylamide gels. It contains two different dyes (xylene cyanol FF and orange G) for visual tracking of DNA migration during electrophoresis. The presence of glycerol ensures that the DNA in the ladder and sample forms a layer at the bottom of the well. The EDTA included in the solutions binds divalent metal ions and inhibits metal-dependent nucleases.

6X Orange DNA Loading Dye is used for conventional DNA electrophoresis (see recommendations for use).

Quality Control

Tested for DNA sample preparation prior to agarose gel electrophoresis. The absence of deoxyribonucleases confirmed by appropriate tests.

Composition

• 10 mM Tris-HCl (pH 7.6),
• 0.15% orange G,
• 0.03% xylene cyanol FF,
• 60% glycerol,
• 60 mM EDTA

Storage

Store at room temperature or at 4°C up to 12 months.
For longer periods, store at -20°C.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Preparation of DNA Samples for Denaturing Polyacrylamide/Urea Gel Electrophoresis

Note

Use the same loading dye solution for the sample and the ladder DNA.

1. Mix the DNA sample with an equal volume of 2X RNA Loading Dye.
2. Heat at 95°C for 5 min.
3. Chill the sample on ice for 3 min.
4. Keep samples on ice while loading.

Non-denaturing PAGE

1. For a nondenaturing 5% polyacrylamide gel solution of 40 ml, mix the following:

   10X TBE Buffer	4 ml
   20% acrylamide/bisacrylamide	10 ml
   Deionized water	26 ml

   Caution: acrylamide is a neurotoxin; always wear gloves, safety glasses, and a surgical mask when working with acrylamide powder.
2. Vigorously agitate the solution for 1 min by magnetic stirring to ensure complete mixing.
3. Add 48 µl of TEMED and swirl the flask to ensure that the solution is thoroughly mixed.
4. Immediately add 240 µl of fresh 10% (w/v) APS and mix thoroughly.
5. Pour the acrylamide between the gel plates and insert the comb.
   Clamp the comb in place at the top of the gel to avoid separation of the gel from the plates as the acrylamide polymerizes. Allow the gel to polymerize for 30 min.
   Important note: polymerization begins as soon as APS is added to the mixture, so all subsequent steps must be performed quickly.
6. After polymerization is complete, remove the comb and any bottom spacers from the gel. Wash the gel plates to clean any spilled acrylamide and be sure that the spacers are properly seated and clean. Fill the lower reservoir of the electrophoresis tank with 1X TBE buffer. Initially, place the gel into the lower tank at an angle to avoid the formation of air bubbles between the plates and the gel bottom. Clamp the gel plates to the top of the electrophoresis tank and fill the upper reservoir with 1X TBE so that the wells are covered.
7. Pre-run and warm the gel for at least 30 min at 5 V/cm (constant voltage).
   Load the recommended volume of the ladder, premixed with the appropriate electrophoresis loading dye solution. Use the same loading dye for the sample DNA.
8. Run the gel at 5 V/cm, taking care to avoid excessive heating. Run the gel for the time indicated in the certificate of analysis of the ladder.
9. Stain the gel in a 0.5 µg/ml ethidium bromide aqueous solution for about 30 min. Examine the gel under the UV light.

Denaturing Polyacrylamide/Urea Gel Electrophoresis

Note

Double stranded DNA ladders are not recommended for denaturing electrophoresis as they may form an atypical pattern. However these usual discrepancies are normally acceptable for analysis of cDNA or other ssDNA in denaturing PAGE.

1. For a denaturing 10% polyacrylamide gel solution of 40 ml, mix the following:

   10X TBE Buffer	4 ml
   20% acrylamide/bisacrylamide	10 ml
   UREA	19.2 g (to 8 M final concentration)
   Deionized water	to 40 ml

   Caution: acrylamide is a neurotoxin; always wear gloves, safety glasses, and a surgical mask when working with acrylamide powder.
2. Vigorously agitate the solution by magnetic stirring to ensure complete mixing and solving of UREA powder.
3. Add 40 µl TEMED and swirl the flask to ensure thorough mixing.
4. Immediately add 400 µl of fresh 10% (w/v) APS and mix thoroughly.
5. Pour the acrylamide between the gel plates and insert the comb.
6. Clamp the comb in place at the top of the gel to avoid separation of the gel from the plates as the acrylamide polymerizes. Allow the gel to polymerize for 30 min.
   Important note: polymerization begins as soon as APS is added to the mixture, so all succeeding actions must be performed promptly.
7. After polymerization is complete, remove the comb and any bottom spacers from the gel. Fill the lower reservoir of the electrophoresis tank with 1X TBE buffer. Initially, place the gel into the lower tank at an angle to avoid the formation of air bubbles between the plates and the gel bottom. Clamp the gel plates to the top of the electrophoresis tank and fill the upper reservoir with 1X TBE so that the wells are covered.
8. Pre-run and warm the gel for at least 30 min at 5 V/cm (constant voltage).
   

   Note

   Heat the gel (buffer) during the whole run at 60-70°C.
9. Wash the wells with 1X TBE buffer to remove UREA and gel pieces.
10. Load the samples.
11. Run the gel at 6 V/cm till the lower dye front reaches the three thirds of the gel.
12. Soak the gel for about 15 min in 1X TBE to remove the urea prior to staining.
13. Stain the gel in a 0.5 µg/ml ethidium bromide aqueous solution for about 30 min.
14. Examine the gel under the UV light.