Agarose gel electrophoresis

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problems/questions

 Problem list:

  • metal rack for storage gels: #Cat.No


Mol.biology // Next-generation sequencing

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Содержание


[править] Gel concentration

dsDNA and dyes in agarose gels of different concentration

[править] Linear molecules

agarose [%]DNA [kbp]
0.3%5-60
0.5%1-30
0.6%1-20
0.7%0.8-12
0.8%0.6-10
0.9%0.5-8
1.0%0.5-7
1.2%0.4-6
1.5%0.2-3
2.0%0.1-2
  • low limit is determined by diffusion of DNA-band in the gel. NB! There are special "
  • upper limit also depends on the voltage: low voltage helps to resolve long fragments



[править] Circular molecules: supercoiled and relaxed

  • relative mobility of "supercoiled" and "circular relaxed" DNA relative to "linear DNA" molecules strongly depends on electrophoresis conditions: agarose concentration and voltage. It is IMPOSSIBLE to use one type of molecules as a mobility marker for another type. Table gives some impression about relative mobility for medium (~6V/cm) and high (~12V/cm) voltage.
supercoiled DNA [kbp]linear DNA [kbp] in different gels
0.7%1%1.5%2%
21.21.31.3 (1.6)1.5 (1.0)
31.71.82 (2.4)2.9 (1.8)
42.22.32.7 (3.7)-
52.72.93.5 (5.5)-
63.23.55 (8.5)-
73.94.28.5 (>12)-
84.45.0>12-
95.15.9--
105.86.8--
1278.7--
  • use low-concentrated gels for preparation of digested vector, it helps to resolve linear and circular molecules
  • to increase mobility difference (in ~20x) of supercoiled and relaxed circular molecules, use:
» 4x TAE, 0.5µg/ml EtBr
» or 1x TAE, but lower concentration of EtBr



[править] Single-stranded DNA (ssDNA)

  • normally, on 1% agarose ssDNA runs ~10% faster, than dsDNA of the same size
  • EtBr staining: 4-5x more (by weight) of ssDNA is necessary to obtain the band of the same brightnes (if compare with dsDNA)
  • two methods of denaturing of "linear" or "nicked circular" DNA just before electrophoresis:
» by heat:
NB! procedure works for <100µl aliquots
100°C, 1min. (in boiling water)
immediately put in ice bath
» by alkaly:
add to DNA NaOH to 0.1M
incubate 1-5min. at room temperature
(optional) neutralize {HCl, 0.1M with TrisHCl, pH7, 0.1M}



[править] Chambers, plates and combs

plateagarose [ml]
soft lid from 96-well plate~90
plate 11x11cm2110-140
plate ??????


  • there are two types of electrophoresis chambers:
» with two platinum electrodes: possible to run with any polarity
» with one platinum electrode: "+" and "-" are strictly defined
  • chamber (spacers, combs) preparation:
▫ wash by sponge with detergent
▫ rinze ~10 times with running water
▫ rinze 2-3 times with bidistillate
▫ dry:
chamber: inverted on paper towel
spacers, combs: blot with paper towel


  • NB!, easy ways to destroy chamber:
» tear off electrode during washing
» snap off the wall of the chamber if hold by the wall only



[править] Preparation of agarose gel

» it is more practicall to prepare a lot of gels in advance and store then in fridge;
» we use metal rack for storage: #Cat.No

  1. prepare agarose:
    • on balance:
    ▫ weight agarose in the conical (Erlenmeyer) flask: do not tare the balance after weighting
    ▫ pour 1x electrophoresis buffer to desired volume
    ▫ tare the balance with flask
    ▫ do not switch off the balance (to add water after boiling)
    NB! Handle hot agarose with caution, it can boil suddenly
    • boil agarose in microwave oven for 1-3min[1]
    • put flask back on the balance, add water to "0"-weight
    • put flask on rocking platform, wait until temperature drops down to 50-60°C[2][3]
  2. prepare plates:
    • prepare a place on the table: wash, blot with paper towel
    • wash and blot plates, spacers, combs
    • assemble plates with combs
  3. pour gels[4]:
    • (for assembled plates) pour ~2ml of agarose on spacers, wait until gel harden (2-4min.)
    • pour agarose, wait until gel harden completely (0.5-1h)
  4. storage
    NB! remove comb from <1% gel just before use
    » leave on the table for 2-4h
    » store in electroforesis chamber under the buffer for days
    » wrap plate with gel and comb in Saran wrap and store for 1-2 months at 4°C



[править] Low-percentage gels (~0.3%)

» gel should be prepared in cold room
» it wold be better to run electrophoresis in cold room
» 0.3% gel is very fragile, always keep it on agarose support, do not pour the buffer on the gel surface
» low-percentage gels are sensitive to overloading


  1. in standard plate prepare supporting gel: 1.5-2.0%, ~2-3mm thick;
  2. fix comb on supporting gel: gap between comb and supporting gel should be ~1-2mm;
  3. pour 0.3% agarose;
  4. remove comb just before electrophoresis under the electrophoresis buffer (otherwise wells would collapse)



[править] Loading

  • sample volume should correspond to the well:
» too low volume results in overloading of the gel
» too low volume of the marker prevents estimation of inclination of NA bands
» too much: part of the sample will be over the upper surface of the gel
  • for fragile agarose (low melting; high resolution) it worth to check wells for leakage. Use 0.5x loading buffer for it (sample will be under this buffer during loading).


[править] Loading buffer

  • universal loading buffer, 10x, store at room temperature. SDS is partially precipirates from the solution, mix buffer before aliquoting.

conc.stock1ml25ml
SDS0.5%10%50µl1.25ml
EDTA, pH8.00.1M0.5M0.2ml5.0ml
Glycerol50%100%0.5ml
0.625g
12.5ml
15.63g
H2O mQ0.25ml6.25ml

» glycerol is for increasing of the solution density
» SDS and EDTA stop any enzymatic reactions


[править] Loading dyes

  • loading buffer is colored:
advantagesdisadvantages
  • color helps for loading
  • dye migrates during electrophoresis and helps to estimate where is the sample in the gel
  • may interfere with NA detection under short-wave light
  • may co-elute from the gel with NA-sample and interfere with downstream applications
  • loading dyes:
DyeStockWork conc.Comments
Xylene Cyanol and Bromph. Blue1% in H2O~0.005-0.02%
  • may interfere with NA detection under short-wave light
  • co-elution in PEG does not interfere with labeling, ligation and transformation
Cresol red (Na salt)50mM in H2O~0.1mM
  • practically does not interfere with NA detection under short-wave light
  • dye is compatible with enzymatic reactions
OrangeG1% in formamide~0.01-0.05%
  • fluorescent under short-wave light
  • have high mobility, practically always below the "working area"
  • mobility of dyes in agarose gel:
agarose gel [%]mobility of loading dye [kbp]
Xylene CyanolCresol RedBromph. BlueOrangeG
0.7%~10.5 (9-15)~3.8 (3.2-4.5)~0.8 (0.7-1.2)~0.15 (0.1-0.2)
0.8%~8 (7-9)~2.9 (2.7-3.2)~0.5 (0.4-0.6)<0.25
1.0%~6 (4.8-7)~2.2 (2-2.5)~0.5 (0.4-0.6)<0.25
1.5%~2.2 (1.8-2.6)~1.0 (0.9-1.3)~0.25<<0.25
2.0%~0.75 (0.6-0.9)~0.35 (0.3-0.4)<0.25<<0.25
  • the only function of dyes is to be visible in well and in the gel. Usually ~0.025% is recommended for Xylene Cyanol and Bromph. Blue. It is too much:
» dye bands occupy large area
» fragments, co-migrating with dye are invisible
  • it is convenient to have on the bench:
» loading buffers with different dyes
» several dilutions of loading buffer: 10x, 2x, 1x. Then, sample may be combined from two components (buffer + sample) instead of three (buffer + sample + water):
▫ 1x if sample volume is < < 20%
▫ 2x ⇒ 20-60%;
▫ 10x ⇒ >60%



[править] Gel capacity

  • low limit is determined by detection method
  • upper limit. Too much DNA in the well have several bad effects:
» smearing of DNA band
» DNA migrates faster. So, do not panic immediately, if marge band of pDNA moves faster, than diluted control plasmid (NB! the same effect when there are a lot of RNA in the DNA sample)
» underestimation of DNA quantity starting from ~0.5µg per dsDNA band per 5mm lane. Optimal range for DNA quantitation is 0.02-0.15µg per 5mm lane.



[править] Voltage

High electric field intensity decrease the "useful length" of the gel
  • for estimation of electric field intensity people measure the distance between electrodes and ignore chamber geometry
  • best way of running the gel: ~6V/cm for the first 5 min[5], ~1V/cm for the next 25 min[6], then normal run: 2-6V/cm.
  • DNA lose EtBr at high temperature. If gel is heated during electrophoresis it may result in fading of bands
  • DNA moves from "-" to "+". EtBr — in the opposite direction. To prevent loosing EtBr from the gel it worth to include dye in the electrophoresis buffer.
  • high electric field intensity
advantagesdisadvantages
  • fast run
  • small diffusion of short fragments
  • decrease of the "effective gel length"
  • heating of the gel and chamber (as a result — bleaching of DNA bands, melting of agarose)
  • more sensitive to overloading
  • for electrophoresis in 1x TAE buffer:
field intensitypurpose of run
<2V/cmovernight
~2V/cmhigh quality and preparative
2-6V/cmanalytical
>6V/cmexpress analysis



[править] Detection

  • the most common dye used to make NA visible for agarose gel electrophoresis is ethidium bromide (EtBr). EtBr fluorescens depends on intercalation into NA. EtBr staining:
» requires >10ng dsDNA per 5mm lane
» 4-5x more (by weight) of ssDNA is necessary to obtain the band of the same brightness (if compare with dsDNA)
  • even short exposure of nucleic acids to UV light reduce significantly the efficiency of PCR and cloning. The best to avoid exposure to UV light is to use blue light excitation source


NB! Normally, there is no restriction for using photo-documentation devices. All of them should be handled as "dirty" (contaminated by other NA) devices


  • it is better (and easier) instead of trying to prepare well-balanced photo to make two photos of the gel:
» underexposed, to see strong bands in details
» overexposed, to make visible faint bands




  1. it is important to boil agarose for some time to melt all agarose granules
  2. at this temperature it is possible to hold flask in hands
  3. it is possible to accelerate cooling:
    » shake bottle in water bath (or under water stream)
    » wrap rocking flask with wet paper towel
    » put rocking platform in cold room
  4. gels would harden faster, if poured in cold room
  5. push NA fragments into agarose
  6. beginning of the electrophoresis is a most dangerous for overloading. It is worth to run in under low voltage


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