Download OLSON LAB PROTOCOL: Agarose Gel Electrophoresis using GelRed

Gel Electrophoresis / Page 1 of 3
OLSON LAB PROTOCOL: Agarose Gel Electrophoresis using GelRed
Revision: 1 / Date: 11.06.2013 / by: S Zafrin / Modified by: PD Olson
NOTES
• Gel electrophoresis is a method used to size fractionate, visualise and document DNA
samples, such as those resulting from PCR
• As DNA molecules are negatively charged, a current can be applied to a gel to draw the
fragments toward the cathode (positive pole)
• Larger fragments move more slowly through the agarose gel matrix than small fragments
and hence the fragments are size fractionated
• The resolution of separation depends on the type and concentration of gel and the length
of the run: ‚mini‘ gels are fairly low resolution and typically used for separating fragments
that differ by >100 bp
• By adding a fluorescent marker that is incorporated into the DNA, the fragments can be
visualised as bands under fluorescent light
• Traditional DNA intercalating markers such as ethidium bromide have been replaced by
less toxic substitutes such as GelRed, which is highly similar to EtBr in structure
• Rough estimates of product concentration can also be made by comparison with the
brightness of products of known DNA quanty, such as the molecular weight marker
• N.B. Agarose and reagents for eletrophoresis are freely available in the labs, and
GelRed and Hyperladders I & IV are available to purchase internally
• N.B. TAE running buffer in the gel tanks can be reused many times, albeit it well
gradually become more concentrated due to evaporation and will change in pH.
Best to replace the running buffer after every 5-10 runs
1. For 1% agarose, add 1 g powdered agarose and 6 ul GelRed to 100 ml 1X TAE buffer
(small mini gels typically require ~50 ml of agarose). Use a labelled 250 ml screw cap
bottle and add a magnetic stir bar
2. Make certain bottle top is loose and melt the agarose gel in a microwave on high power
for 40-60+ sec, then cool while stirring with a magnetic stir bar
3. Choose a gel tray and comb(s) combination sufficient for the number and volume of
samples plus one or more molecular weight markers (i.e. Hyperladder)
4. Tape the ends of the gel tray using heat-resistant tape such as autoclave indicating tape
and insert the comb(s)
5. Pour the agar into the gel tray, making the gel thick enough only to provide sufficient
space in the wells to hold the sample volumes. NB. thinner gels make the bands easier to
visualise
6. Let the gel set fully before using and carefully remove the comb to avoid tearing the wells
7. Make sure the gel tank is filled with TAE buffer (ie. enough to cover the top of the gel).
NB. use of water as a running buffer or in the gel mix will result in bizarre smears
and interpretable gels!
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8. Choose an appropriate molecular weight marker (aka ‘ladder’) for the expected PCR
product sizes. Hyperladder I includes bands between 200 and 10,000 bp
9. Load 5 ul of the appropriate Hyperladder molecular weight marker in the first well (and
last if wanted). NB. If 5 ul of Hyperladder are loaded, then each band in the ladder
will represent a known DNA concentration in ng (see Hyperladder images at
bottom)
10. Cut a square of Parafilm and on it dot 4 ul of loading buffer for each sample, then add 1
ul of PCR product (NB. change tips throughout!). Note that the loading buffer contains
one or more tracking dyes that can be seen to migrate through the gel and provides
enough density for the PCR sample to sink in the wells.
11. Mix the 1 ul of sample and 4 ul of loading buffer by gently pipetting up and down and then
load the entire 5 ul into a well. If necessary, place the gel tank over a dark surface to aid
in visualizing the wells.
12. Put the cover on the gel tank making sure the gel is oriented such that the wells are
closest to the negative pole. NB. if you accidentally reverse the polarity your samples
will be run out of the gel and into the buffer and be lost
13. Connect the black (negative) and red (positive) leads to a power pack and set the voltage
between 80 and 100
14. Set the timer on the power pack depending on the size of the gel and distance you wish
to run the products through the gel. For mini gels, 30-40 mins is usually sufficient
15. Carry the gel in the tray to the gel documentation system (NB. be careful that it doesn’t
slide onto the floor!) to visualise the gel under fluorescent light. If the bands are not
separately sufficiently, return to gel tank and continuing running
16. Use the gel documentation system to photograph the results and print and annotate a
photo in your lab book
TROUBLESHOOTING
Note that while gel electrophoresis is simple and routine, it is prone to producing artefacts
such as shadow bands, and can thus often be misleading. Thus it is used primarily as a
quick estimate of PCR product fidelity, size and quantity. Below are some common problems
with gels generally; see PCR Amplification for problems caused during PCR reactions.
No bands whatsoever, including the ladder
• GelRed or ethidium bromide not added to gel or samples
• Samples and/or ladder not added to loading buffer
No bands, only smears, including the ladder
• Water used instead of TAE buffer in the gel and/or running buffer: always use 1X TAE for
both making and running the gels.
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Bands are fuzzy, fat and/or hard to focus
• Comb set at an angle: remake gel ensuring comb is straight up and down
• Gel is too thick making it more opaque: use a thinner gel
• Gel run too fast: reduce operating voltage to 70-90
• Gel concentration too low: try using 1.5%-2% agarose
Bands are angled, or form a smile shape
• Current not running evenly through the gel: current will find the path of least resistance,
thus if gel tray is narrower than gel tank, the current will run more quickly through the
running buffer than through the gel, sometimes creating a gradient of current across the
gel. Make sure tray is placed centrally or use a gel tray that spans the full width of the
tank
• Gel consistency uneven: fully melt, mix and allow gel to cool slowly in order to produce
an even consistency
• Gel was physically damaged: make new gel
Ladder bands look OK, but PCR bands fuzzy, smeary, feint, etc.
• Problem lies with the PCR reaction: see the Protocol for PCR Amplification
Wells of gel fluoresce brightly
• High molecular weight DNA is stuck in the wells: if visualising a DNA extraction, this is a
good indication the gDNA is of good quality (not fragmented). If visualising a PCR
product, this generally indicates that excess template was used: reduce template amount
by 1-3 ul