Agarose gel electrophoresis of DNA fragments Page 1 of 5
(Maniatis, Sambrook, BioWhittaker catalogue)
Method: DNA in solution has a net negative charge due to its phosphate backbone (at the pH used during electrophoresis). Therefore, molecules with different lengths can be separated upon electrophoresis through a porous matrix. Agarose is used as a matrix for fragments from 100 base pairs through entire genomes of several megabases. Acrylamide is used for sequencing of DNA and separation of short stretches of DNA (oligonucleotides). Ethidium bromide (EtBr) or Roti®-GelStain can be added to visualize the DNA when the gel is exposed to UV light. For standard cloning (and subsequent Southern blotting) the following protocol for horizontal agarose electrophoresis is mostly used.
We use the one glove policy read the document “working with Ethidium bromide”
General protocol:
• Add agarose (0.7 - 2 %) in 1X electrophoresis buffer (1X TAE or 0.5X TBE) • Dissolve the agarose by heating the solution (i.e. with microwave) • Be aware of solution that is coming out of Erlenmeyer. • When the solution is clear (i.e. agarose melted) cool the solution to ‘hand warm’ or place it at 60°C • Add 5 µl Roti-GelStain to 100 ml agarose solution ‚ mix and pour onto gelling surface with a ‘comb’ inserted.
• When the solution has fully gelled, the comb can be removed and the gel placed in the electrophoresis apparatus • Add buffer to the electrophoresis apparatus, such that the buffer over the gel is 2 to 3 mm • Apply the DNA samples 5 µl ) mixed with 1X Orange-G loading buffer in the wells produced by the comb.
Add the right marker • Run the gel (1V/cm => better separation - 10V/cm => faster separation) until the Orange dye has reached the end of the gel • The DNA (actually nucleic acid dyes intercalated with the DNA) can be visualized using UV-irradiation • Careful: UV is harmful to the skin and eyes • The gel can be used for Southern blotting or isolation of the DNA fragments
Agarose_electrophoresis.docx 6 april 2018
Agarose gel electrophoresis of DNA fragments Page 2 of 5
Preparing agarose gels
Microwave method: • Use a 250 ml bottle with maximal 150 ml 1X TAE-buffer • Add agarose to buffer solution while stirring (1.5%) • Put the flask with unscrewed cap in the microwave • Heat the solution (setting high) for 1 min • Gently swirl the flask to dissolve any powder and gel pieces • Be careful: the solution can become ‘superheated’ and foam over when agitated • Reheat in the microwave until a homogenous solution is obtained • Swirl to mix and cool to ~60oC • Pour the gel in a cassette or gel tray and position the comb • Wait for gelling to finish (~30min)
Solutions:
50X TAE: 0.4 M Tris (242.0 g/l), 0.2 M Acetic Acid glacial (57.1 ml/l), 20 mM Na2EDTA.2H2O (37.2 g/l), Check pH (=7.7) and adjust volume to 1000 ml with dH2O
1X TAE Mix 20 ml 50X TAE with 980 ml H2O
10X TBE: 0.9 M Tris (215 g/2l), 0.9 M boric acid (110 g/2l), 25 mM EDTA (2H2O) (18.6 g/2l), pH 8.3
EtBr stock solution: o 1 mg/ml in H2O (store at 4 C in a dark bottle)
Roti®-GelStain: Carl-Roth 3865 (1ml solution)
6X loading buffer (for 10 ml , in water): 1ml 1M Tris pH8.0, 3ml 100% Glycerol (or 2.5g Ficoll or 4g sucrose), 25mg bromophenol blue or 15 mg Orange G
Agarose_electrophoresis.docx 6 april 2018
Agarose gel electrophoresis of DNA fragments Page 3 of 5
REMARKS Agarose related remarks: • Agarose loses its gel strength noticeably 5 years after production. • Avoid agents that disrupt hydrogen bond formation (e.g. urea), since that will decrease melting temperature, gelling temperature and gel strength. • Make sure that all agarose is dissolved before pouring the gel. A non-homogenous gel (in agarose concentration) will result in strange shaped bands. • Don’t pour the agarose solution when >60oC. The gelling surface may bend or even crack. • Dried agarose on the comb teeth can fracture the agarose upon comb removal and result in distortion of the resulting DNA bands. • Low melting temperature agarose and low percentage agarose gels are especially sensitive to agarose fracturing. To prevent agarose fracturing upon comb removal cool gel to 4oC or place gel under cold buffer before comb removal. • Loose gel fragments should be removed from the wells by flushing the wells prior to loading the gel to prevent loading problems.
Sample related remarks: - Samples containing ethanol (or other compounds lowering the density of the sample) can sometimes ‘come out of’ the well.
Buffer related remarks: • A buffer is added because during electrophoresis water is electrolyzed such that near the cathode the solution becomes basic and near the anode the solution becomes acidic. • TAE doesn’t buffer as good as TBE, but gives a better separation in the high molecular weight region (>12 kb). Use for recovering DNA from the gel. • TBE gives a better separation in the low molecular weight region (<1 kb), but precipitates easily upon storage • Buffer depletion will cause gel melting, smearing of DNA and/or overheating. Recirculating the buffer can help prevent these problems.
DNA mobility remarks: • Nucleic acid dye does have an effect on the mobility of DNA (~15% reduced). If this interferes with your application, one can run an agarose gel without dye and stain the gel after electrophoresis. • Circular fragments (i.e. plasmids) will mostly give 3 fragments on gel, due to the occurrence of supercoiled DNA (‘covalently closed’, smaller volume than linear DNA), nicked DNA (‘open closed’, slightly smaller volume than linear) and linear DNA. The ‘open closed’ and linear forms will occur mostly due to mechanical/chemical breaking of one (‘open closed’) or both strands (linear) of the DNA during isolation. • Bromophenol Blue and other dyes generally used to monitor electrophoresis (Xylene Cyanol) do not share the same mobility characteristics as DNA. Therefore, the fragments of DNA with which they co-migrate are different with different buffers, agarose concentrations and even agarose types.
Agarose_electrophoresis.docx 6 april 2018
Agarose gel electrophoresis of DNA fragments Page 4 of 5
DNA size % agarose Up to 60 kb 0.3% 1000 bp – 30 kb 0.5% 800 bp – 12 kb 0.7% 500 bp - 10 kb 1.0% 400 bp – 7 kb 1.2% 200 – 3000 bp 1.5% 50 - 2000 bp 2.0%
Dye mobility remarks: Generally used dyes are bromophenol blue (BFB) and Xylene Cyanol (XC). Dye migration (when compared to dsDNA) in agarose depends, besides the obvious (% agarose) also on the quality of the agarose and the used buffer. Therefore take caution in used the following table.
Dye migration in agarose gels (in bp) in 1 X TAE in 1 X TBE % agarose BFB XC BFB XC 0.3 2900 24800 2850 19400 0.5 1650 11000 1350 12000 0.75 1000 10200 720 9200 1.0 500 6100 400 4100 1.25 370 3560 260 2500 1.5 300 2800 200 1800 1.75 200 1800 110 1100 2.0 150 1300 70 850
Resolution related remarks: - A 3 to 4 mm thick gel gives best resolution (for horizontal gels) - Glycerol (up to 20%) can be added to prevent diffusion of low molecular weight fragments (<0.5Md). - Thickness of the comb in the direction of the electric field affects resolution. A thin comb will give sharper DNA bands. - Glycerol in the loading buffer allows DNA to stream up the sides of the well before electrophoresis is started, resulting in a U-shaped band. The higher molecular weight Ficoll prevents this. - A high ionic strength in the loading buffer can result in ‘fuzzy’ bands. Best is to use a loading buffer with the same (or lower) ionic strength of the electrophoresis buffer. - Best resolution is obtained with 4-10 V/cm (cm = distance between the electrodes, not gel length). Lower voltages increases diffusion of small fragments, while higher voltages increases band streaking of >12kb fragments.
Agarose_electrophoresis.docx 6 april 2018
Agarose gel electrophoresis of DNA fragments Page 5 of 5
DNA detection in gels: Different fluorescent (DNA binding) compounds are used to detect DNA in gels. ssDNA and RNA can also be detected, but the sensitivity is lower.
Compound detection limit excitation l emission l Ethidium bromide 0.2ng 302, 505 nm 602 nm SYBR green I 60pg 494, 284, 382 nm 521 nm SYBR green II 60pg 497, 254 nm 520 nm Gelstar 60pg 493 nm 527 nm Roti-GelStain 0.2ng 310, 540 nm 630 nm
Post-run-staining:
One glove policy.
For optimal resolution, lowest background and uniform staining however, it is best to stain the gel following electrophoresis. - After electrophoresis (without Dye added) submerge the gel in 5 µl/100 ml Dye (in water or electrophoresis buffer) for 20 min Ethidium bromide is a powerful mutagen. Avoid skin contact. This solution can be re-used several times. - Replace the Dye solution for distilled water or buffer (2 x 20 min) - Check the results upon exposure to UV light UV light is harmful. Avoid exposure to skin and eye. - Continue incubation with distilled water or buffer until a satisfactory result is obtained
Agarose_electrophoresis.docx 6 april 2018