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Electrophoresis of nucleic acids

Gel type selection Maintenance Gel loading Run electrophoresis NA visualization NA recovery from gels Markers Solutions  Comments

Gel type selection

Nondenaturating separation

• very large fragments (up to 5Mb): pulse-field electrophoresis;



• relatively large dsDNA fragments: normal agarose

  Agarose [%] Separation range [kb] Tracking dye migration [kb] Xylene cyanol FF Cresol red Bromphenol blue Orange G 0.3 5-60   always below DNA fragments 0.5 1-30   0.6 1-20 10-13 4-5 0.9-1.5 0.7 0.8-12 9-15 3.5-4.5 0.7-1.2 0.8 0.6-10 7-9 2.7-3.5 0.6-1.1 0.9 0.5-8 6-8 2.5-3.2 0.6-1 1.0 0.5-7 5-7 2-2.5 0.5-0.8 1.2 0.4-6 4-5 1.8-2 0.4-0.7 1.5 0.2-3 1.8-2.6 0.9-1.3 ~0.25 2.0 0.2-2 0.6-0.9 0.3-0.4 < 0.25




• 50bp-2kbp dsDNA fragments: 2% high-resolution agarose;



• relatively small dsDNA fragments: nondenaturating PAAG

  PAA [%] Separation range [bp] Tracking dye migration [bp] Xylene cyanol FF Bromphenol blue Orange G 3.5 1000-2000 460 100 always below DNA fragments 5.0 80-500 260 65 8.0 60-350 160 45 12.0 40-200 70 20 15.0 25-150 60 15 20.0 6-100 45 12





Denaturating separation

• rough single-strand separation of "complex" NA mixture: denature NA just before loading or use denaturaling loading buffer. This method does not work for mixtures of low complexity, because they renature again too fast.



• relatively large DNA fragments: denaturating agarose gel;



• relatively small DNA fragments: denaturating PAAG

  PAA [%] Separation range [bp] Tracking dye migration [bp] Xylene cyanol FF Bromphenol blue Orange G 4 100-500 230 50 always below DNA fragments 5 70-300 130 35 6 45-70 105 26 7 40-60 88 22 8 35-45 75 19 10 25-35 55 12 20 8-25 28 8 30 2-8 20 6

Maintenance

Plastic

electrophoresis chamber, agarose plates, spacers, combs, gel-pouring apparatus, etc.
• normal washing:
  • wash with detergent (use different sponges for "pre-PCR" and "post-PCR" plastic);
  • rinse 3-5 times with tape water;
  • rinse 2-3 times with bidistilled water;
• to remove RNases: incubate (rinse) with 2% H₂O₂ for 15-30 min, then normal washing;
• to remove DNA: incubate (rinse) in 10% bleach for 15-30 min, then normal washing;
• drying spacers & combs: paper towel or 100% ethanol;





Glass and titanium oxide plates

• normal washing:
  • wash with detergent (use different sponges for "pre-PCR" and "post-PCR" plates);
  • rinse 3-5 times with tape water;
  • rinse 2-3 times with bidistilled water;
• to remove bind-silane-attached gel: incubate in 0.5M KOH (NaOH) for ~30 min, then normal washing;
• durty plates: incubate (rinse) in 1M KOH in methanol for 15-30 min, then normal washing;
• washing of organic, marker: acetone;



• siliconising:
  • be sure, that plate is dry (100% ethanol wash accelerate drying),;
  • distribute few drops of Repel-Silane using dust-free paper & polish untill plate is completely dry;
  • repeat previous step;
  • rinse plate with 70% ethanol;
  
  
  
  • one siliconization is enough for several gel preparations.

Gel loading

• Two components of loading buffer are obligatory: tracking dyes and high density component. Others (ptional) components can (i) preserve buffer at room temperature, (ii) stops enzymatic reactions, (iii) inhibir DNase activity, etc.



• To accelerate work it is better to have on the bench buffers of different concentration (10x, 2x, 1x) to combine sample from two components (sample, buffer) instead ot three (sample, water, buffer). Buffers should be with individual tracking dyes. Multiple tracking dyes may be combined them from individual components befor use.




Tracking dyes

• help to insert sample into well; (ii) help to control electrophoresis process. But they can interfere with visualization of NA fragments.

  Dye stock working concentration notes [x] [%] Xylene cyanol FF   Cresol red   Bromphenol blue   Bromocresol green for alkaline agarose gel electrophoresis Orange G 1% in formamide always runs below NA fragments






High-density component

• help to load sample into well

  component working concentration Sucrose 40%(w/v) in 6x buffer Ficoll (Type 400) 15%(w/v) in 6x buffer Glycerol 30%(w/v) in 6x buffer 50%(w/v) in 10x buffer Formamide 95%(w/v) in 2x buffer






Loading on the gel

• denaturing PAAG: high-density urea diffuse in the wells during pre-electrophoresis. It is necessary to wash it out before loading;
• to make wells clear visible use black background or LED-lamp with flexsible stand;
• be accurate not to create the bridge between surface of the buffer and the sample if high-molecular weight DNA or detergent is present in loading buffer, it will result in moving sample onto the surface. Move tip out of the well under the surface of the buffer;
• to check integrity of the wells load on the gel portions of loading bffer without sample (actual for shark-tooth combs and for high-resolution agarose gels);
• to separate samples from each other on shark-tooth sequencing gel load first all odd-samples, run them into the gel, then load even samples and run electrophoresis;
• try do not load samples simmetrically on the gel (especially PAAG);
• do not put too low volumes in the wells, because: (i) sample distribution is uneven along the band; (ii) for agarose gels effective area of the gel will be lower (overloading, different mobility of samples and markers);
• do not put too much volumes in the wells, because: (i) well leakage; (ii) agarose gel forms menisque nearby with a comb, so material from the upper part of the well would not enter the gel;
• maximum loading capacity depends on the type of electrophoresis:
  • high-resolution agarose: ~0.12µg/mm² (3µg per normal well);
  • normal agarose: ~0.24µg/mm² (6µg per normal well);
  • non-denaturating PAA:
  • denaturating PAA: ~2µg/mm²;

Run electrophoresis

Electric field strength

• measurement:
  • vertical gels: voltage/lenght of the gel;
  • horizontal gel: voltage/distance between electrodes in chamber;
• too low electric field results:
  • unacceptably long run;
  • diffusion of bands;
• too high electric field results:
  
  • shortening of effective length of the gel (long fragments run relatively faster);
  • low resolution of long fragments;
  • overloading starts early;
  • gel overheating (depending on temperature: removal of NA dyes, gel smiling, faster diffusion, band distortion, band inclination, agarose melting);





Pre-electrophoresis

• not necessaryy for agarose gels;
• 10-15 min for polyacrylamide gels. This time it not enough to heat the gel up to working temperature. Use hot buffers, hot metal plate, 50°C incubator.





Temperature

• diffusion is faster at high temperature. It is important for small fragments, large fragments diffuse too slowly, other factors are more important for them;
• double-stranded fragments >50bp are not dissociate in denaturing urea gel untill temperature reach 50-60°C. It is better to run denaturing gels at ~50°C.
• Electrophoretic mobility increase with temperature. Higher temperature of central part of verlical gel lead to "smiling" of NA bands. Metal heat-distribution plates or upper bufer contacting plate are used to prevent smiling.





Electro-geometry

• agarose gels:
  • electric field strength determined by geometry of electric chamber only, gel does not influence it;
  • to minimize heating keep minimal buffer depth over the gel;
• vertical acrylamide gels:
  • electric field strength determinedonly by the length of the gel (width and thiknes have no effect);
  • when thikness increase: (i) maximum loading amount per well proportionally increase; (ii) heating increase; (iii) band resolution decrease;
  • when gel width increase: (i) number of wells proportionally increase; (ii) temperature of the gel does not change; (iii) band resolution does not change;
  • to slow down mobility of short fragments: (i) use gels with gradient of concentration; (ii) use gradient spacers (wider on the bottom); (iii) use lower buffer with higher ionic strength;

NA visualization

UV shadows

nucleic acids strongly adsorb UV light. If put the gel on fluorescent surface and shine it with 260-280nm UV lump dark regions uppear under the bands.

• does not work with thick (agarose) gels;
• sensitivity is ~0.1µg per 3mm band;
• normal office paper covered with Saran-wrap is a nice fluorescent surface;





Ethidium bromide (EtBr)

Fluorescent yeld of EtBr is ~20—30-fold greater if it binds to DNA. Normal working concentration is 0.5µg/ml.
• stock solution: 10mg/ml, 20 000x, store at NT in dark;
• UV_250-280 is adsorbed by DNA and transmitted to dye, UV_300-380 adsorbed directly by dye (~4 times less sensitivity). Re-emission is at 590nm (red-orange);
• ~10ng DNA band may be visible without dye washing;
• EtBr moves in opposite to NA direction during phoresis (worth to include it in lower buffer);
• EtBr may be used in buffers with urea, glyoxal, formamide, formaldehyde;
• EtBr stains oligos and RNA, but ~5 times less efficient if compare with dsDNA (seems, it stains small ds-regions; staining of oligos is very sequence-specific);
• EtBr is stable in aquatious solution, EtBr-DNA complex is stable at NT;
• staining:
  • EtBr may be included in agarose gel (and buffer) in advance;
  • EtBr inhibit acrylamide polymerisation and shuld not be included into PAA mix;
  • EtBr does not bind DNA in alkaline solutions (alkaline gels should be stained after electrophoresis);
  • may be included into sample before loading on the gel (ovegheating of the gel results in destaining);
  • may be included into lower buffer and will enter the gel during electrophoresis;
  • gel may be stained after electrophoresis;
• EtBr is potential mutagen and cancerogen (but was used previously as a human and nowdays as a cattle antitrypanocidal drug);





SYBR dyes

SYBR Gold, SYBR Green I, SYBR Green II. About 20-100 times more sensitive then EtBr.

stain specificity pH optimum spectrum [nm] notes excitation emission SYBR Gold ds and ss NA 7.0-8.5 300, 495 537 prestain not recomended, because of high retardation SYBR Green I dsDNA 7.5-8.0 (preferably 8.0) 290, 380, 497 520 may be included into gel in advance SYBR Green II ssDNA, oligos, RNA 254, 497 520




• stock solution: 10 000x in DMSO, store desiccated, in dark at -20°C;
• relatively expensive, use minimal staining volume;
• may be used in buffers with urea, glyoxal, formamide, formaldehyde. Use plastic container for staining, do not use glass;
• unstable in aquatious solutions, SYBR-NA complexes sre stable at NT for some time;
• moves in opposite to NA direction during phoresis;
• staining:
  • dye may be included into sample before loading on the gel: (i) distort electrophoretic mobility, (ii) lower sensitivity, (iii) ovegheating of the gel results in destaining;
  • potential mutagens and cancerogens;





GelRed & GelGreen

About 20-100 times more sensitive then EtBr. Stable in water solutions.
• Adsorbtion at 250-300 and around 500nm for both. Re-emission is at 540nm (green) or 600nm (red);
• stock solution: 10 000x (3 300x for post staining) in DMSO or water, store at NT in dark;
• may induce retardation of NA;





UV / Blue light sources

• 260nm: highest sensitivity for EtBr staining; guite harmfull for NA;
• 312nm: still high sensitivity for EtBr staining; less dangerous for NA; suitable for SYBR staining;
• 380nm: ~5x lower sensitivity if compare with EtBr staining; much less dangerous for NA; suitable for SYBR staining;
• Blue light (420—500nm): does not damage NA, suitable for GYBR- and GEL- staining;



• it is nice to have in the lab:
  • hand-held illuminator for: (i) control of electrophoresis; (ii) shadow imaging and (iii) band cutting;
  • transilluminator & camera for gel documentation;





Isotope labeling

Intensively labeled thin gels may be exposed without any treatment wrapped in Saran-wrap. For long exposure it is necessary fix and dry gel.
• thin gel may be fixated by freezing: wrap into Saran-wrap; expose gel in precooled (-20°C) cassete;
• denaturing PAAG:
  • fixation in {10% acetic acid, 10% ethanol} untill tracking dyes would practically washed: ~5 min for 0.2mm gel, ~15 min for 0.4mm, ~1 hour for 1mm;
  • drying on 3MM (gel dryer) or covalently bounded to the glass (hair dryer);
• agarose gels (neutral and alkaliy):
  • soak the gel in 7% TCA 30 min at NT;
  • dry using paper towelor gel dryer or hair dryer;





Silver staining

NA recovery from gels

• agarose gel:
  • silica adsorbtion (Qiagen spin columns, glass milk);
  • organic extraction from low melting point agarose;
  • electroelution into dialysis bag;
  • electrophoresis onto DEAE-cellulose membrane;
  • crush-and-soak;
  • elution into PEG/TAE;



• PAAG — only crush-and-soak procedure;



• If NA band is too large (wide range of fragments should be isolated) it is possible to put it in reverse orientation into a new gel of the same composition and run electrophoresis again. Thin band will form.



• It is necessary to minimize UV-damage during band excision.

Markers

• Sometimes markers (even from a good suppliers) have a bad quality: they looks diffusive on the gel. It seems, that the reason is a high concentration of salt in the marker.



• Single-stranded DNA runs ~10% faster, then dsDNA of the same size.



• Mobility of circular and supercoiled DNA strongly depend on electrophoresis conditions: concentration of agarose, electric field strength, etc. It is impossible to use linear DNA as a marker.

  Supercoiled DNA [kbp] Linear DNA [kbp] in different concentration of agarose gel [%] at different voltage: 6V/cm (12V/cm) 0.7% 1% 1.5% 2% 2 1.2 1.3 1.3 (1.6) 1.5 (1.0) 3 1.7 1.8 2 (2.4) 2.9 (1.8) 4 2.2 2.3 2.7 (3.7) - 5 2.7 2.9 3.5 (5.5) - 6 3.2 3.5 5 (8.5) - 7 3.9 4.2 8.5 (>12) - 8 4.4 5.0 >12 - 9 5.1 5.9 - - 10 5.8 6.8 - - 12 7 8.7 - -

Solutions

TAE, 50x

Tris-Acetate Electrophoresis (TAE) buffer, 1x pH=7.6, ρ_50x=1.0878g/ml
store large quantities at 4°C, working amount at NT

10x 1x stock 100ml 150ml 200ml 250ml Tris-base 40mM 2M 121.1g/M 24.22g 36.33g 48.44g 60.55g EDTA 1mM 0.05M 372.3g/M 1.862g 2.792g 3.723g 4.654g Acetic acid ~30mM ~1.56M 17.4M ~8.96ml ~9.40g ~13.44ml ~14.10g ~17.92ml ~18.80g ~22.40ml ~23.50g ~ ~ H2O   mQ ~73.30g ~109.95g ~146.60g ~183.25g ~





TAE, 1x

stable at NT for couple of weeks

conc. stock 1L 1.5L 2L TAE 1x 50x 20ml / 21.76g 30ml / 32.63g 40ml / 43.51g / EtBr 0.5µg/ml 10mg/ml 50µl 75µl 100µl H2O   mQ 0.98L 1.47L 1.96L





TBE, 10x

Tris-Borate Electrophoresis (TBE) buffer, 1x pH=8.3, ρ_10x=1.07g/ml
store filtered at NT (precipitetes in durty bottle)

1x 10x stock 1L Tris base 89mM 0.89M 121.14g/M 107.8g Boric acid 89mM 0.89M 61.83g/M 55.03g EDTA, pH 8.0 2mM 20mM 500mM 40ml 43.84g H2O   mQ 863.3ml

• 10x stock solution is stable at NT after 0.4µm filtration;
• for PAAG use as 1x, for agarose gel — as 0.5x;
• TBE is used in agarose gels for separation of small (< 200bp) fragments. Purification of fragments from TBE-based gels generally is more complicated than from TAE-based gels.




TBE, 1x

stable at NT for couple of weeks

conc. stock 1L 1.5L 2L TBE 1x 10x 100ml / 107g 150ml / 160.5g 200ml / 214g / EtBr 0.5µg/ml 10mg/ml 50µl 75µl 100µl H2O   mQ 0.9L 1.35L 1.8L





FGRB, 50x

Formaldehyde Gel-Running Buffer (FGRB), 1x pH=7.0, ρ_50x=1.104g/ml
store at +4°C

1x 50x stock 0.5L 1.0L 2.0L MOPS (pH7.0) 20mM 1.0M 209.27g/M 104.64g 209.27g 418.54g AcONax3H2O 10mM 0.5M 136.08g/M 34.02g 68.04g 136.08g EDTAx2H2O 1mM 50mM 372.3g/M 9.31g 18.62g 37.23g H2O   mQ 404.0ml 808ml 1616ml




50x stock 0.5L 1.0L 2.0L MOPS (pH7.0) 1.0M 209.27g/M 104.64g 209.27g 418.54g AcONa anhydrous 0.5M 82.04g/M 20.51g 41.02g 82.04g EDTA x 2H2O 50mM 372.3g/M 9.31g 18.62g 37.23g H2O   mQ 417.5ml 835ml 1670ml

• adjust pH7.0 10N NaOH (per 500ml of buffer ~22ml), filter-sterilize through 0.22µm filter;
• solution becomes yellow during the storage on the light, but it has no effect on the solution quality.




GTGB, 20x

Glycerol Tolerant Gel Buffer (GTGB), ρ_20x=1.094g/ml
store at +4°C

1x 20x stock 1L 1.5L 2.0L 2.5L Tris base 89mM 1.78M 121.14g/M 216g 324g 432g 540g Taurine 28mM 0.56M 125.15g/M 72g 108g 144g 180g Na2EDTAx2H2O 0.53mM 10.7mM 372.3 g/M 4g 6g 8g 10g H2O   mQ 802ml 1203ml 1604ml 2005ml





Loading buffer, 10x

10x, ((none) NT).

10x stock 1ml 5ml 10ml 25ml Xilene cyanol 0.1% solid 1.0mg 5.0mg 10mg 25.0mg Bromph. blue 0.1% solid 1.0mg 5.0mg 10mg 25.0mg SDS 0.5% 10% 50µl 250µl 500µl 1.25ml EDTA, pH8.0 0.1M 0.5M 0.2ml 1.0ml 2.0ml 5.0ml Glycerol 50% 100% 0.5ml 0.625g 2.5ml 3.125g 5.0ml 6.25g 12.5ml 15.63g H2O   mQ 0.25ml 1.25ml 2.5ml 6.25ml





Formamide 2x loading buffer

Formamide with one or several tracking dyes. May be used as 1-2x buffer. Normally, commercial formamide have acceptable quality, but if it is yellow, it should be deionised.