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In vitro Transcription

10X TBE Buffer (Tris-borate-EDTA)

Product information

Applications

• Electrophoresis of nucleic acids in agarose and polyacrylamide gels.
• Used both as a running buffer and as a gel preparation buffer.
• Filtered through a 0.22 µm membrane.
• Recommended for electrophoresis of RNA and DNA fragments smaller than 1500 b(p).

1X Composition

• 89 mM Tris,
• 89 mM boric acid,
• 2 mM EDTA,
• pH of 10X TBE: 8.3

Usage Recommendations

• Use fresh 1X TBE both for the gel and for the electrophoresis run.
• To prepare 1X TBE buffer, add 100 ml of 10X TBE buffer to 900 ml of deionized water and mix well.

Quality Control

The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.

Note

Double-stranded linear nucleic acid molecules migrate about 10% slower in TBE buffer than in TAE buffer.

Storage

There are no time limitations for storage of the electrophoresis buffers at room temperature. If the buffer is stored at lower temperatures, a precipitate may form, which is easily dissolved by gentle heating.

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Preparation of DNA Ladders/Markers for Electrophoresis

Table 1. Recommendations for loading the conventional formulation (supplied in TE buffer) DNA ladders/markers.

Technical specifications	Conventional formulation (supplied in TE buffer) DNA ladders/markers
	GeneRuler™ DNA ladders	Lambda DNA markers	Phage & Plasmid DNA markers	Markers for Genomic DNA Analysis
Supplied amount / number of applic.	50 µg (100 µl) is sufficient for: 100 applic. on agarose gel 50 applic. on native PAGE	50 µg (100 µl) is sufficient for 100 applic. on agarose gel	50 µg (100 µl) is sufficient for: 100 applic. on agarose gel 50 applic. on native PAGE	6 µg (30 µl) is sufficient for 120 applic. on agarose gel
Amount used per 1 mm width of a gel lane	0.1 µg (0.2 µl) for agarose gel 0.2 µg (0.4 µl) for PAGE	0.1 µg (0.2 µl) for agarose gel	0.1 µg (0.2 µl) for agarose gel 0.2 µg (0.4 µl) for PAGE	6 ng
Dilution	Not needed	Not needed	Not needed	Mix 1 µl (0.2 µg) of DNA marker with 39 µl of nuclease-free water
Heating	Do not heat	Heat at 65°C for 5 min; chill on ice for 3 min before use	Do not heat	Heat at 65°C for 5 min; chill on ice for 3 min before use
I. Loading on agarose gel:
DNA ladder/marker loading dye Water, nuclease-free	1 µl (0.5 µg) 2 µl 9 µl	1 µl (0.5 µg) 2 µl 9 µl	1 µl (0.5 µg) 2 µl 9 µl	10 µl (50 ng) of diluted marker 1 µl
Mix gently and load on gel
II. Loading on polyacrylamide gel:
DNA ladder/marker loading dye Water, nuclease-free	2 µl (1 µg) 0.5 µl 0.5 µl	Not recommended for PAGE	2 µl (1 µg) 0.5 µl 0.5 µl	Not recommended for PAGE
Mix gently and load on gel

Table 2. Recommendations for loading ready-to-use DNA ladders/markers.

Technical specifications	DNA ladders/markers, ready-to-use
	GeneRuler™ & O'GeneRuler™ DNA ladders	MassRuler™ DNA ladders	FastRuler™ DNA ladders	O'RangeRuler™ DNA ladders	ZipRuler™ Express DNA ladders	Lambda DNA markers	Phage & Plasmid DNA markers
Supplied volume/number of applic.	500 µl for 100 applic.	2x500 µl for 50-200 applic.	2x500 µl for 50-333 applic.	500 µl for 100 applic.	2x500 µl for 100-200 applic.	500 µl for 100 applic.	500 µl for 100 applic.
Heating	Do not heat					Heat at 65°C for 5 min; chill on ice for 3 min before use	Do not heat
Mix gently and load on gel
Volume per 1 mm width of a gel lane	1-2 µl	variable	variable	1-2 µl	1-2 µl	1-2 µl	1-2 µl

Preparation of DNA Samples for Conventional DNA Electrophoresis

6X DNA Loading Dye, 6X MassRuler™ DNA Loading Dye, 6X Orange DNA Loading Dye, 6X TriTrack™ DNA Loading Dye are all used according to below protocol:

1. Add 1 volume of 6X DNA loading dye to 5 volumes of DNA sample.
2. Mix well, spin down and load.

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 Gels in TBE Buffer

1. Prepare 20 ml of a 5% polyacrylamide gel containing 7 M urea by adding:

   47.5% acrylamide: 2.5% bis-acrylamide solution	2 ml
   10 M urea	14 ml
   10X TBE Buffer	2 ml
   10% freshly prepared ammonium persulfate	0.2 ml
   Deionized water	1.8 ml

2. Mix and add 10 µl TEMED. Mix again and pour the gel carefully avoiding the formation of air bubbles.
3. Insert the comb into the acrylamide and allow the gel to polymerize for at least 1 hour.
4. Fill the electrophoresis apparatus with 1X TBE buffer.
5. Heat the RNA samples and ladder at 70°C for 10 min, and chill on ice for 3 min.
6. Load onto the gel.
7. Run electrophoresis at 8 V/cm for about 1 hour.
8. Soak the gel for about 15 minutes in 1X TBE to remove urea prior to staining.
9. Stain the gel in 0.5 µg/ml ethidium bromide in 1X TBE solution for 15 min.