Optimal concentration between 0.4-5% in all typical buffer systems.
GQ (Genetic Quality) certified, which ensures that nucleic acids recovered from preparative gels can be used for downstream applications (enzymatic reactions etc.).
Low DNA/RNA binding.
Excellent gel transparency.
DNase and RNase free.
Applications
Analytical electrophoresis of nucleic acids.
Preparative electrophoresis.
Blotting assays.
Characteristics
Electroendosmosis EEO – 0.05-0.13 Gel strength (1% gel) – >1200 g/cm2 Gel strength (1.5% gel) – >2500 g/cm2 Gelling temperature – 36±1.5°C Melting temperature – 88±1.5°C Moisture – <7%
Quality Control
The absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests.
Figure 1. Separation of Fermentas DNA Ladders in TopVision™ LE GQ Agarose. Electrophoresis conditions: 1% gel, 0.5 µg/lane, 1X TAE, 7V/cm, 40 min. 1 – GeneRuler™ DNA Ladder Mix, ready-to-use 2 – GeneRuler™ 1kb DNA Ladder, ready-to-use 3 – GeneRuler™ 1kb Plus DNA Ladder, ready-to-use 4 – ZipRuler™ Express DNA Ladder 1, ready-to-use 5 – ZipRuler™ Express DNA Ladder 2, ready-to-use 6 – GeneRuler™ Express DNA Ladder, ready-to-use
Figure 2. Separation of Fermentas RiboRuler™ RNA Ladders in TopVision™ LE GQ Agarose. Electrophoresis conditions: 1X TAE buffer, 7 V/cm, 40 min. 1 – RiboRuler™ Low Range RNA Ladder, 2 µl/lane, 2% gel 2 – RiboRuler™ High Range RNA Ladder, 2 µl/lane, 1% gel
Perform electrophoresis of DNA in low melting point (LM) agarose (#R0801) gel prepared in TAE (#B49), 0.5X TBE, TBE (#B52) or TPE buffer. Stain the gel with ethidium bromide.
Cut out the desired DNA band from the agarose gel with a clean scalpel under UV light*. Cut out only as much agarose as it is necessary. (The bottom of the excised agarose is free of DNA and should be removed.)
Determine the weight of the slice. To facilitate melting, cut gel slices larger than 200 mg into smaller pieces.
Incubate the tube at 70°C for approx. 10 min. Ensure that the agarose is completely melted.
Transfer the tube to a 42°C water bath and equilibrate for 5 min.
Add 1 u of Agarase (#EO0461) per 100 mg (approx. 100 µl) of molten 1% low melting agarose. Increase the amount of enzyme proportionally for higher percentage agarose, gently mix and incubate at 42°C for 30 min.
Add ammonium acetate** to a 2.5 M final concentration, chill on ice for 5 min.
Centrifuge at 10,000 rpm for 10 min to pellet undigested carbohydrates. Transfer the supernatant to a clean tube.
Add 2.5 volumes of ethanol or 0.8 volume of isopropanol, mix gently and incubate at room temperature for 1 h. If DNA fragments are smaller than 500 bp or if the DNA concentration is lower than 0.05 µg/ml, incubate at room temperature for 2 h.
Centrifuge at 10,000 rpm for 15 min, remove supernatant and dry the pellet. Resuspend the pellet in TE or another appropriate buffer for subsequent manipulation.
Note
* For subcloning of gel-purified DNA fragments, care should be taken to avoid DNA damage with UV light. Minimize the UV exposure to a few seconds or keep the gel on glass or plastic plate during UV illumination. Alternatively, visible dyes can be included in standard agarose gels to visualize DNA bands in ambient light (1, 2). ** Ammonium acetate, is recommended rather than other salts to avoid co-precipitation of oligosaccharides with DNA.
The procedure typically recovers 90% of DNA from the gel.
For evaluation of DNA yield use Fermentas DNA ladders/markers, which are ideal for in-gel DNA quantification.
T4 polynucleotide kinase is inhibited by ammonium ions, therefore use 1 M Sodium Acetate, (0.3 M final concentration) if T4 polynucleotide kinase will be used in downstream applications.
Large DNA fragments (>30 kb) require delicate handling to avoid mechanical shearing. After digestion with agarase (step 6), centrifuge at maximum speed for 10 min to pellet undigested carbohydrates. Remove oligosaccharides and agarase by dialysis or carry out subsequent manipulations with DNA in the digested agarose solution.
References
Rand, K.N., Crystal Violet can be used to Visualize DNA Bands during Gel Electrophoresis and to Improve Cloning Efficiency, Elsevier Trends Journals Technical Tips Online, T40022, 1996. 2. Adkins, S., Burmeister, M., Visualization of DNA in agarose gels and educational demonstrations, Anal. Biochem., 240 (1), 17-23, 1996.
General Recommendations for DNA Electrophoresis
Use the same DNA loading dye (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
If possible, always load equal volumes of the sample DNA and the ladder/marker DNA. The sample can be diluted with 1X DNA loading dye.
Avoid high salt concentrations in the DNA samples as this may cause bands to shift during electrophoresis.
Following electrophoresis, visualize DNA by staining in 0.5 µg/ml ethidium bromide solution or SYBR® Green I.
Choose the gel percentage according to the tables below: Table 1. Recommended Agarose Gels for Electrophoretic Separation of DNA Fragments.
Agarose gel, %
Range of effective separation, bp
Approximate positions of tracking dyes, bp*
Bromophenol blue
Xylene cyanol FF
TBE buffer
TAE buffer
TBE buffer
TAE buffer
0.5
2000-50000
750
1150
13000
16700
0.6
1000-20000
540
850
8820
11600
0.7
800-12000
410
660
6400
8500
0.8
800-10000
320
530
4830
6500
0.9
600-10000
260
440
3770
5140
1.0
400-8000
220
370
3030
4160
1.2
300-7000
160
275
2070
2890
1.5
200-3000
110
190
1300
1840
2.0
100-2000
65
120
710
1040
3.0
25-1000
30
60
300
460
4.0
10-500
18
40
170
260
5.0
10-300
12
27
105
165
Table 2. Recommended Polyacrylamide Gels for Electrophoretic Separation of DNA Fragments (1).
Polyacrylamide gel (with BIS at 1:20), % (w/v)
Range of effective separation*
Approximate positions of tracking dyes*
Bromophenol blue
Xylene cyanol FF
Denaturing gels
4.0
100-500 b
50 b
230 b
5.0
70-400 b
35 b
130 b
6.0
40-300 b
26 b
105 b
8.0
30-200 b
19 b
75 b
10.0
20-100 b
12 b
55 b
15.0
10-50 b
10 b
0 b
20.0
5-30 b
8 b
28 b
30.0
1-10 b
6 b
20 b
Non-denaturing gels
3.5
100-1000 bp
100 bp
460 bp
5.0
80-500 bp
65 bp
260 bp
8.0
60-400 bp
45 bp
160 bp
12.0
50-200 bp
20 bp
70 bp
15.0
25-150 bp
15 bp
60 bp
20.0
5-100 bp
12 bp
45 bp
Note
* Positions of the tracking dyes can only be estimated approximately because the dye front migrates as wide band. The following guidelines are recommended:
Only high purity agarose should be used. TopVision™ Agarose was used to prepare the gels.
Only freshly prepared electrophoresis buffers should be used. The buffers were prepared from Fermentas 50X TAE Buffer and 10X TBE Buffer.
Choose electrophoresis conditions according to the recommendations below:
Size of the DNA
Voltage
Buffer
<1 kb
5-10 V/cm
TBE
1-5 kb
4-10 V/cm
TAE or TBE
> 5 kb
1-3 V/cm
TAE
Up to 10 kb, fast electrophoresis with Express DNA ladders
up to 23 V/cm
TAE
Recommendations for Accurate Gel Quantification
Always use the same DNA loading dye (supplied with the DNA ladder/marker) for both the sample DNA and the ladder/marker DNA.
Always compare the sample band with the ladder band of the closest size.
If possible, adjust the concentration of the sample to approximately equalize it with the amount of DNA in the nearest band.
dNTPs, oligonucleotides, genomic DNA, RNA, NTPs or buffer components can interfere with spectrophotometrical measurements and lead to inaccurate quantification of sample DNA. In these cases, it is best to rely on gel quantification data.
For the most accurate quantification, use video-densitometry analysis.
Reference
Sambrook, J., et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 12.89, 5.42, 2001.
General Recommendations for RNA Electrophoresis
RNA ladders, as well as any RNA, are extremely sensitive to degradation by ribonucleases. Use only fresh electrophoresis buffers and freshly poured gels.
Use clean electrophoresis chambers. For RNA gel analysis, avoid electrophoresis tanks used for DNA miniprep analysis since DNA minipreps may contain RNase A or T1.
Use the same loading dye for samples and for RNA markers. 2X RNA Loading Dye is available separately and is provided with all RiboRuler™ RNA ladders. It contains ethidium bromide for RNA visualization on denaturing formaldehyde gels. If RNA fragments are separated on native agarose gels or on polyacrylamide/urea gels, additional staining with ethidium bromide is recommended.
For native gels, add 0.5 µg/ml of ethidium bromide to the agarose gel and to the running buffer.
Preparation of RNA Ladders for Electrophoresis
For RiboRuler™ RNA ladders:
Mix 1 volume of RNA ladder and 1 volume of the supplied 2X RNA Loading Dye.
Heat at 70°C for 10 min.
Chill on ice for 3 minutes and spin down prior to loading on a gel.
Load 0.5 µl of the prepared ladder for every mm of gel lane width (4 µl / 8 mm lane). For RiboRuler™ RNA ladders, ready-to-use:
Heat RNA ladders at 70°C for 10 min.
Chill on ice for 3 minutes and spin down prior to loading on a gel.
Load 0.5 µl of the ladder for every mm of gel lane width (4 µl / 8 mm lane).
Note
Ladders prepared as described above are not suitable for glyoxal/DMSO agarose gel electrophoresis.
Non-denaturing Agarose Gel
Use an Erlenmeyer flask of at least three times larger volume than that of the solution to avoid boiling over.
Use the same 1X electrophoresis buffer to prepare the gel and to run electrophoresis.
Dilute 50X TAE or 10X TBE buffers to a 1X concentration immediately before use.
Use TBE buffer for analysis of RNA bands smaller than 1500 b. For larger RNA, use TAE buffer.
Weigh out the required amount of agarose (depending on the gel %) into an Erlenmeyer flask.
Add the appropriate volume of either 1X TBE or 1X TAE buffer and swirl to mix.
Weigh the flask with the solution. For high percentage gels (3-5%):add an excess amount of distilled water to increase the weight by 10-20%.
Boil the mixture in a microwave oven (at middle power) until the agarose melts completely; swirl the flask several times while boiling. To prepare the highest quality agarose gels of any percentage, an additional 3-5 min of boiling after completely melting the agarose is recommended. A significant amount of water evaporates during this procedure and therefore restoring of the initial weight (in step 5) is required to obtain the desired percentage gel.
Weigh the flask again and if necessary, add hot distilled water to restore the initial weight. For high percentage gels (3-5%): check (by weighing) that the excess 10-20% of water has evaporated and, if needed, continue boiling to remove any excess, or add hot distilled water to restore the initial weight. Optional.For intensified gel staining add ethidium bromide to a final concentration of 0.5 µg/ml. Mix well and heat for an 1 minute without boiling.
Cool the solution to 65-70°C. Pour carefully on a clean casting plate with an appropriate comb. If bubbles appear, push them carefully away to the sides with a pipette tip.
Solidify the gel for approximately 30 min before use. Low percentage LM agarose gels can be solidified at 4°C.
Immerse the gel into the desired electrophoresis buffer.
Heat the RNA samples and ladder at 70°C for 10 min, then chill on ice for 3 min. Load onto the gel.
Run electrophoresis at 5 V/cm until the bromophenol blue runs approximately two-thirds of the way down the gel.
After electrophoresis the gel can be stained by immersing it into a 0.5 µg/ml ethidium bromide solution for 20 min, stained with SYBR® Green II or any other RNA staining technique. Warning. Hot agarose solution should be handled very carefully.
Denaturing Formaldehyde Gels in MOPS Buffer
Freshly prepare 10X MOPS buffer: 0.4 M MOPS (pH 7.0), 0.1 M sodium acetate, 0.01 M EDTA (pH 8.0).
Prepare 1% TopVision™ Agarose gel as follows:
stir 1g of agarose powder in 72 ml of deionized water;
melt the agarose, and then add 10 ml of 10X MOPS buffer and mix;
when the agarose solution cools to 60°C, add 18 ml of fresh formaldehyde (37%) in a fume hood and mix thoroughly;
pour the gel.
Place the gel into an electrophoresis apparatus containing 1X MOPS buffer.
Heat the RNA samples and ladder at 70°C for 10 min, and then chill on ice for 3 min.
Load onto the gel.
Note
There is no need to stain the gel as ethidium bromide present in 2X RNA Loading Dye is sufficient for visualization under UV light.
Non-denaturing PAGE
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.
Vigorously agitate the solution for 1 min by magnetic stirring to ensure complete mixing.
Add 48 µl of TEMED and swirl the flask to ensure that the solution is thoroughly mixed.
Immediately add 240 µl of fresh 10% (w/v) APS and mix thoroughly.
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.
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.
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.
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.
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.
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.
Vigorously agitate the solution by magnetic stirring to ensure complete mixing and solving of UREA powder.
Add 40 µl TEMED and swirl the flask to ensure thorough mixing.
Immediately add 400 µl of fresh 10% (w/v) APS and mix thoroughly.
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 succeeding actions must be performed promptly.
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.
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.
Wash the wells with 1X TBE buffer to remove UREA and gel pieces.
Load the samples.
Run the gel at 6 V/cm till the lower dye front reaches the three thirds of the gel.
Soak the gel for about 15 min in 1X TBE to remove the urea prior to staining.
Stain the gel in a 0.5 µg/ml ethidium bromide aqueous solution for about 30 min.
- Worldwide
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- Store at room temperature
Figure 1. Separation of Fermentas DNA Ladders in TopVision™ LE GQ Agarose.
Figure 2. Separation of Fermentas RiboRuler™ RNA Ladders in TopVision™ LE GQ Agarose.
