UNIT 7.6
Denaturing Gel Electrophoresis for
Sequencing
The accuracy of DNA sequence determination depends largely upon resolution of the
sequencing products in denaturing polyacrylamide gels. This unit provides a detailed
description of the setup, electrophoresis, and processing of such gels.
In general, the gels required for DNA sequencing are 40-cm long, of uniform thickness,
and contain 4% to 8% acrylamide and 7 M urea (basic protocol). Modifications of the
basic protocol increase the length of readable sequence information which can be obtained
from a single gel. These include forming the gel with wedge-shaped spacers to create a
field gradient (Ansorge and Labeit, 1984; Olsson et al., 1984), or incorporating a buffer
gradient (first alternate protocol), an electrolyte gradient (second alternate protocol), or
an acrylamide step gradient (Isfort and Ihle, 1988; Williams et al., 1986) into the gel. It
is also possible to use longer gels (80 to 100 cm), although these can be technically
challenging to pour and handle.
Another modification to the basic protocol—inclusion of formamide in the sequencing
gel—is designed to overcome gel compressions arising from secondary structure in the
sequencing products during gel electrophoresis (third alternate protocol). Formamide
concentration ranges of 25% (Brown, 1984) to 40% (USB, 1990) can be used to alleviate
gel compressions. See critical parameters and troubleshooting for a discussion of acrylamide concentrations and electrophoresis conditions.
CAUTION: Acrylamide and bisacrylamide are neurotoxins and should be handled with
gloves. Dimethyldichlorosilane should also be handled with gloves and plate treatment
with this solution should be carried out in a fume hood (see silanization, APPENDIX 3).
TEMED and formamide should also be handled with care.
BASIC
PROTOCOL
POURING, RUNNING, AND PROCESSING SEQUENCING GELS
This protocol describes preparing the sequencing plates, pouring the sequencing gels, and
loading, running, and processing the gels to analyze a set of sequencing reactions (UNITS
7.4 & 7.5).
The steps are the same for all acrylamide concentrations; formulations for 4%, 6% and
8% gels are provided in reagents and solutions. Refer to critical parameters for a
discussion of choosing the appropriate acrylamide concentration.
Materials
70% ethanol or isopropanol in squirt bottle
5% dimethyldichlorosilane (diluted in CHCl3; Sigma #D-3879)
Denaturing acrylamide gel solution
TEMED
10% (w/v) ammonium persulfate (made fresh weekly and stored at 4°C)
1× TBE buffer, pH 8.3-8.9 (APPENDIX 2)
Sequencing samples in formamide/dye solution (UNITS 7.4 or 7.5)
5% acetic acid/5% methanol (vol/vol) fixer solution
Denaturing Gel
Electrophoresis
for Sequencing
7.6.1
Supplement 17
30 × 40–cm front and back gel plates
0.2- to 0.4-mm uniform-thickness spacers
Large book-binder clamps
60-ml syringe
Pipet tip rack or stopper
0.2- to 0.4-mm sharkstooth or preformed-well combs
Sequencing gel electrophoresis apparatus
Contributed by Barton E. Slatko and Lisa M. Albright
Current Protocols in Molecular Biology (1992) 7.6.1-7.6.13
Copyright © 2000 by John Wiley & Sons, Inc.
Pasteur pipet or Beral thin stem
Power supply with leads
Sequencing pipet tip
Gel dryer
Shallow fixer tray
46 × 57–cm gel blotting paper (e.g., Whatman 3MM)
Kodak XAR-5 X-ray film
NOTE: Many companies provide equipment needed for sequencing experiments; a list of
suppliers is provided in Table 7.6.1.
Assemble gel sandwiches
1. For each gel, meticulously wash a pair of 30 × 40–cm front and back gel plates with
soap and water; rinse well with deionized water and dry.
A thorough washing and rinsing is critical. Soap residue decreases polymerization time,
thus risking polymerization while pouring the gel. Dust and other particulates (e.g., residue
from previous gels) left on the plates will make it extremely difficult to pour a gel that does
not contain bubbles. Particulates in the gel will also distort the banding pattern.
2. Wet plates with 70% ethanol or isopropanol in a squirt bottle and wipe dry with a
Kimwipe or other lint-free paper towel.
3. Wearing gloves, apply a film of 5% dimethyldichlorosilane in CHCl3 to one side of
each plate by wetting a Kimwipe with the solution and wiping the whole plate
carefully. After the film dries, wipe with 70% ethanol or isopropanol and dry with a
Kimwipe. Check plates a final time for dust and other particulates.
Perform this step in a fume hood.
There are two reasons for silanizing the plates: to facilitate pouring a gel without bubbles
and to prevent the gel from sticking to the plates during postelectrophoresis processing (see
critical parameters for further discussion of silanization strategies).
Table 7.6.1
Suppliers of Sequencing Gel Electrophoresis Equipmenta
Equipment
Supplierb
Sequencing gel apparatus, including
necessary clamps, combs and spacers
Gel tape
AAP, ABA, BR, GB, HO, IBI, JS,
OSP, PH, SS
ABA, GB, HS, IBI, OSP
Power supplies
ABA, ACS, BR, EC, FD, GB, HO,
IBI, IS, OSP, PH, SS, ST
Beral thin stem
Ultrathin pipet tips for loading gels
BE
ABA, BR, CO, DR, DY, HO, IBI, IS,
MB, PH, SS, ST
Gel thermometer
Shallow fixer tray
BR
OSP
Transfer paper
Gel dryers
ABA, SS, WH
ATR, BR, HO, SV
aModified from Slatko,
1991a.
AAP, Ann Arbor Plastics; ABA, American Bioanalytical; ACS, Accurate Chemical and
Scientific; ATR, ATR; BE, Beral Enterprises; BR, Bio-Rad; CO, Costar; DR, Drummond; DY, Dynalab;
EC, EC Apparatus; FD, Fotodyne; GB, Gibco/BRL; HO, Hoefer; IBI, International Biotechnologies; IS,
Integrated Separation Systems; JS, Jordan Scientific; MB, Marsh Biomedical; OSP, Owl Scientific Plastics;
PH, Pharmacia LKB; SS, Schleicher & Schuell; ST, Stratagene; SV, Savant; WH, Whatman. See APPENDIX
4 for addresses of suppliers.
bAbbreviations:
DNA Sequencing
7.6.2
Current Protocols in Molecular Biology
Supplement 16
4. Assemble gel plates according to the manufacturer’s instructions with 0.2- to 0.4-mm
uniform-thickness spacers and large book-binder clamps, making certain the side and
bottom spacers fit tightly together.
A good fit between side and bottom spacers is crucial to prevent leaks. If necessary, use a
small amount of acrylamide or 1% agarose to seal the edges and bottom of the gel.
Alternatively, before the top gel plate is placed on the spacers and bottom plate, put a very
small dab of high-temperature vacuum grease at the point where the bottom spacer meets
the side spacers (the grease can be removed later with 70% ethanol).
Gel sandwiches may also be prepared by using two side spacers only (no bottom spacer)
and taping the plates together along the sides and bottom using yellow vinyl electrical tape.
To prevent leakage, tape the bottom of the sandwich a second time forming “hospital bed
corners.” It is important that the tape make good tight contact along its length with the
sides of the gel plates and that any bubbles are smoothed out.
Prepare gel solution and pour sequencing gel
5. For each gel, prepare 60 ml of the desired denaturing acrylamide gel solution in a
100-ml beaker.
The gel mix can be heated to speed dissolution of the urea; however, to prevent degradation
of the acrylamide, do not heat over 55°C. Allow the solution to cool to room temperature
(≤25°C) to prevent polymerization while pouring the gel.
If particulate matter remains after mixing, filter the solution through Whatman No. 1 filter
paper in a funnel.
6. Thoroughly mix 60 µl TEMED, then 0.6 ml of 10% ammonium persulfate, into each
acrylamide solution. Add these components immediately before pouring each gel
(step 7).
These reagents initiate polymerization and should not be added earlier than this stage. To
achieve slower polymerization (an advantage when learning to pour gels), the amounts of
TEMED and ammonium persulfate can be reduced to 40 ìl and 0.4 ml, respectively.
To check the freshness of the ammonium persulfate solution and TEMED, test the polymerization time. Transfer 0.6 ml of the gel mix to a 1.5-ml microcentrifuge tube and add 0.6
ìl TEMED and 6 ìl of 10% ammonium persulfate. Mix the solution by inverting the tube
several times. Polymerization should occur within 5 min. If it does not, make fresh
ammonium persulfate and test another 0.6 ml sample. The amount of TEMED can also be
adjusted.
7. Pour the gel immediately. Gently pull the acrylamide solution into a 60-ml syringe,
avoiding bubbles. With the short plate on top, raise the top of each gel sandwich to a
45° angle from the benchtop and slowly expel the acrylamide between the plates
along one side. Adjust the angle of the plates so that the gel solution flows slowly
down one side.
Other methods of delivering the gel solution include using a 25-ml pipet or pouring the
solution through the sidearm of a 125-ml sidearm flask. Air bubbles can be prevented by
pouring the gel solution at a steady pace while maintaining constant contact between the
fluid being poured and the fluid already between the plates. Any air bubbles that form can
be removed by tapping the glass plates behind the bubbles or by rocking the plates until
the bubbles move to the top of the gel solution.
Denaturing Gel
Electrophoresis
for Sequencing
8. When the gel solution reaches the top of the short plate, lay the gel sandwich down
so that the top edge is ∼5 cm above the benchtop; place an empty disposable pipet
tip rack or stopper underneath the sandwich to maintain the low angle.
9. Insert the flat side of a 0.2- to 0.4-mm sharkstooth comb into the gel solution 2 to
7.6.3
Supplement 16
Current Protocols in Molecular Biology
3 mm below the top of the short plate, being very careful to avoid bubbles. Use
book-binder clamps to pinch the combs between the plates so that no solidified gel
forms between the combs and the plates. Layer extra acrylamide gel solution onto
the comb to ensure full coverage. Rinse the syringe with water to remove excess
acrylamide.
The flat side of the sharkstooth comb creates a flat surface at the top of the gel. Be careful
not to insert the comb too far into the gel. This will make it difficult to insert the teeth all
the way into the gel when the comb is reversed in step 18. Combs with preformed wells are
also commercially available. If this type of comb is used, insert the teeth 2 to 3 mm below
the top of the short plate after the gel is poured and pinch with book-binder clamps as
described above.
If tape was used to seal the gel sandwiches, clamp the sides of the gel together with
book-binder clamps in the positions where the gel will be clamped to the electrophoresis
apparatus. Place the clamps over the side spacers and not over the gel itself. This minimizes
distortion of the gel in the apparatus (see critical parameters).
10. Check for leaks in the gels and slow or stop them by placing book-binder clamps at
these spots. Observe polymerization of the gel solution.
Polymerization requires 10 min to 2 hr, depending on the temperature of the room (i.e., cold
acrylamide polymerizes more slowly) and the amount of TEMED and ammonium persulfate
added. When polymerization is complete, a Schlieren line will be visible at the border of
the comb and the surface of the gel.
11. Use immediately or store gels ≤48 hr at room temperature.
To store gels, place a paper towel dampened with 1× TBE buffer over the comb. Put entire gel
sandwich in a sealed plastic bag or wrap top of the gel tightly with plastic wrap.
Set up the sequencing gel
12. Remove the bottom spacer or the tape at the bottom of each gel sandwich.
13. Remove extraneous polyacrylamide from around the combs with a razor blade. Clean
spilled urea and acrylamide solution from plate surfaces with water.
This step helps to remove the comb cleanly and prevents particles of polymerized acrylamide from falling onto the gel surface.
14. Remove sharkstooth combs gently from each gel sandwich, avoiding pulling or
stretching the top of the gel. Clean combs with water so they will be ready to be
reinserted in step 18.
If a preformed-well comb was used, take care to prevent tearing of the polyacrylamide
wells. This comb will not be reinserted.
15. Fill the bottom reservoir of each gel apparatus with 1× TBE buffer such that the gel
plates will be submerged 2 to 3 cm in the buffer.
16. Place each gel sandwich in a sequencing electrophoresis apparatus and clamp the
plates to the support per manufacturer’s instructions.
If a bottom spacer was used, a large air bubble will often get trapped at the bottom of the
gel. Place the gel sandwich on one bottom corner in the apparatus and slowly lower the
sandwich to chase out a large portion of this air bubble. Squirt buffer under the plates using
a syringe with a bent 20-G needle to remove any remaining bubbles.
17. Pour 1× TBE buffer into the top reservoir to ∼3 cm above the top of the gel. Rinse
the top of the gel with 1× TBE buffer using a Pasteur pipet or Beral thin stem to
remove fragments of excess polyacrylamide and urea leached from the gel.
Check for leaks from the top buffer tank. Pieces of clay or agarose can be used to seal leaks
DNA Sequencing
7.6.4
Current Protocols in Molecular Biology
Supplement 16
from the top buffer tank gasket. To prevent “smiling” (distortion) of the gel bands, check
that the buffer level is horizontal and parallel to the bottom of the gel (see critical
parameters/troubleshooting).
18. Reinsert the teeth of the cleaned sharkstooth combs into the gel sandwich with the
points just barely sticking into the gel. Using a Pasteur pipet or Beral thin stem, rinse
the wells thoroughly with 1× TBE buffer to remove stray fragments of polyacrylamide.
19. Preheat the gels by turning on the power supplies to 45 V/cm, 1700 V, 70 W constant
power ∼30 min before loading sequencing samples.
Each commercial gel apparatus will have its own recommendations about voltage and
power settings. In general, power settings should be 45 W to 70 W with constant power.
Load and run the gel
20. Rinse wells just prior to loading the gels to remove urea that has leached into them.
21. Cover and heat completed sequencing samples in formamide/dye solution for 2 min
at 95°C, then place on ice.
22. Load 2 to 3 µl of each sample per well on each gel to be run. Rinse sequencing pipet
tip twice in the lower reservoir after dispensing from each reaction tube.
It is helpful to mark the outside glass plate with individual lanes or sets of four lanes to
keep track of the loading order.
Sample loading using ultrathin DNA sequencing pipet tips is recommended (Table 7.6.1).
With practice, all samples can easily be loaded in ≤5 min, eliminating the need for
electrophoresing one set of samples into the gel before loading the next set.
23. Run gels at 45 to 70 W constant power according to the manufacturer’s recommendations. Maintain a gel temperature of ∼65°C.
Temperatures higher than this can result in cracked plates or smeared bands, while too low
a temperature can lead to incomplete denaturation of the sequencing products. Monitor
the temperature by taping a round-faced gel thermometer with a flat back surface (Table
7.6.1) to the glass plate with paper tape. Dab a spot of vacuum grease between the glass
plate and the thermometer to assist in heat conduction.
24. Observe the migration of the marker dyes (Table 7.6.2) to determine the length of the
electrophoresis.
If sequencing reactions are loaded on multiple gels, the gels must be coordinated such that
sequences in common from the long and short runs will allow overlapping of the sequence,
yet still maximize the amount of sequence obtained from the long run (see critical
parameters/troubleshooting for examples of electrophoresis times).
Table 7.6.2 Migration of Oligodeoxynucleotides (Bases) in
Denaturing Polyacrylamide Gels in Relation to Dye Markers
Polyacrylamide (%)
Bromphenol blue
Xylene cyanol
5
6
8
10
35 b
26 b
19 b
12 b
130 b
106 b
75 b
55 b
Denaturing Gel
Electrophoresis
for Sequencing
7.6.5
Supplement 16
Current Protocols in Molecular Biology
Process and dry the gel
Many mishaps occur when processing sequencing gels because they are very thin and
fragile and can easily be torn. Minimal handling is essential in the following steps.
25. Fill the dry ice traps attached to the gel dryer (if required) and preheat the dryer to
80°C.
26. After electrophoresis of each gel is complete, drain the buffer from the upper and
lower reservoirs of the sequencing apparatus and discard the liquid as radioactive
waste.
If two gels are run for a set of sequencing reactions, the difference in electrophoresis times
allows processing and drying of each gel as soon as it is finished.
27. Remove the gel sandwich from the electrophoresis apparatus and place under cold
running tap water until the surfaces of both glass plates are cool.
This facilitates handling of the gels and prevents the gels from “curling” as they cool.
28. Lay the sandwich flat on paper towels with the notched (short) plate up. Remove
excess liquid and remaining clamps or tape.
29. Remove one side spacer and insert a long metal spatula between the glass plates where
the spacer had been. Pry the plates apart with a gentle rocking motion of the spatula.
The gel should stick to the bottom plate. If it sticks to the top plate, flip the sandwich
over. Slowly lift the top plate from the side with the inserted spatula, gradually
increasing the angle until the top plate is completely separated from the gel.
If the gel sticks to the top plate in an isolated spot, a stream of water from a squirt bottle
can be sprayed at the spot to aid separation. Should gels recurrently stick to both glass
plates during processing, more thorough cleaning of the glass plates prior to electrophoresis and/or resilanization of the plates may be necessary.
30. Once the plates are separated, remove the second side spacer and any extraneous bits
of polyacrylamide around the gel.
31. If samples contain 32P, the fixing steps (31 and 32) are optional; proceed to step 33.
If samples contain 35S, transfer the gel on the glass plate to a shallow fixer tray and
gently cover to a depth of 2 cm with 5% acetic acid/5% methanol fixer solution. Soak
the gel 10 to 15 min. Gently rock the tray periodically to gradually loosen the gel
from the glass plate.
Urea quenches the 35S signal and must be removed from 35S-containing gels as described
here. Although not required, removing the urea will also increase the clarity of 32P-containing gels.
Occasionally, one or two spots on the gel will stubbornly stick to the glass plate. In this
case, free the gel from the plate by moving it gently with a gloved hand. Extreme care must
be taken at this stage to avoid stretching or tearing the gel.
A tray with a hole in the bottom through which the fix solution can be drained is useful
(Table 7.6.1).
32. Reposition the gel over the plate and remove the fixer solution by aspiration or gravity.
Take care to keep the gel centered over the glass plate at this point. Carefully lift the
plate with the gel on top from the tray.
33. Place gel on a benchtop. Hold two pieces of dry blotting paper together as one piece.
Beginning at one end of the gel and working slowly towards the other, lay the paper
on top of the gel. Take care to prevent air bubbles from forming between the paper
and the gel.
DNA Sequencing
7.6.6
Current Protocols in Molecular Biology
Supplement 16
34. Peel the blotting paper up off the plate—the gel should come with it. Gradually curl
the paper and gel away from the plate as it is being pulled away.
Whatman 3MM seems to work best for this procedure.
Optional: this method is more reliable but slightly more tedious. Set another glass plate on
top of Schleicher & Schuell #GB002 blotting paper and center it over the gel. Flip the entire
sandwich over so that the gel is now resting on top of the blotting paper. Position the
sandwich with the glass plates extended ∼10 cm past the edge of the benchtop. Slowly slide
only the bottom plate back onto the benchtop. Without the support of the bottom plate, the
weight of the gel and blotting paper causes them to slowly peel away from the top plate.
Reposition the sandwich so that the bottom glass plates extend ∼10 cm over the edge of the
benchtop (the top glass plate is now extended ∼20 cm over the edge) and repeat the process.
Lift the top plate from the portion of the gel still in contact with it. If the gel persists in
sticking to the top plate, use a stream of water from a squirt bottle to loosen the gel.
35. Place the paper and gel on the preheated gel dryer. Cover with plastic wrap.
Remove any bubbles between the plastic wrap and the gel by gently rubbing the covered
surface of the gel from the middle toward the edges with a Kimwipe.
36. Dry the gel thoroughly 20 min to 1 hr at 80°C. Peel the plastic wrap away.
The time required for drying depends on the efficiency of the dryer vacuum. When the gel
is completely dry, the plastic will easily peel off without sticking. The plastic wrap will
absorb 35S emissions and must be removed prior to autoradiography. Autoradiography
without plastic wrap will improve the clarity of sequencing ladders from reactions containing 32P.
If a gel dryer is not available, place the paper and gel on a glass plate. Clip the paper to
the plate to prevent curling. Dry in a forced-air oven or overnight at room temperature.
37. Place each dried gel in a separate X-ray cassette with Kodak XAR-5 film in direct
contact with the gel and autoradiograph at room temperature.
Autoradiography is described in APPENDIX 3. No intensifying screen is used. Equivalent
films from other manufacturers can be used.
If necessary, several sheets of paper towels or filter paper can be layered in the cassette
before enclosing the dried gel and X-ray film. This ensures that the X-ray film and gel are
tightly abutted during exposure in the X-ray cassette. Take care not to overpack the cassette
which may cause light leaks around the cassette edges.
38. After sufficient exposure time (usually overnight), remove the X-ray film and process
according to manufacturer’s instructions.
ALTERNATE
PROTOCOL
BUFFER-GRADIENT SEQUENCING GELS
Increasing the ionic strength in a sequencing gel decreases the voltage gradient and
therefore decreases the rate of migration of DNA fragments (Biggin et al., 1983). In this
protocol, the sequencing gel contains a higher concentration of buffer at the bottom of the
gel than at the top and as a result, the migration of shorter oligodeoxynucleotides (at the
bottom) are slowed down relative to the longer oligodeoxynucleotides (at the top). This
allows the gel to be run longer without losing the shorter oligodeoxynucleotides off the
bottom and improves the resolution between longer oligodeoxynucleotides.
Additional Materials
Denaturing Gel
Electrophoresis
for Sequencing
Buffer-gradient gel solutions (containing 0.5× and 2.5× TBE; see reagents and
solutions)
25-ml pipet equipped with a rubber pipet-filler bulb
7.6.7
Supplement 16
Current Protocols in Molecular Biology
1. Assemble one gel plate sandwich as in steps 1 to 4 of basic protocol.
2. In two 100-ml beakers, prepare buffer-gradient gel solutions: 50 ml using 0.5× TBE
(clear solution) and 25 ml using 2.5× TBE (blue solution). Heat gently at ≤50°C while
stirring until all the solids have dissolved. Cool the solutions until they are room
temperature (≤25°C).
3. Add 20 µl TEMED and 200 µl of 10% ammonium persulfate to the 0.5× TBE/gel
solution and mix gently.
4. Add 10 µl TEMED and 100 µl of 10% ammonium persulfate to the 2.5× TBE/gel
solution and mix gently.
5. Using a 25-ml pipet equipped with a rubber pipet-filler bulb, pull up 12.5 ml of the clear
0.5× TBE/gel solution followed gently by 12.5 ml of the blue 2.5× TBE/gel solution.
6. Allow three or four air bubbles to be taken up into the pipet by gently squeezing the
suction inlet on the rubber bulb.
This causes the two layers to gently mix at the interface.
7. Release the solution down the glass plate sandwich in a gentle, even manner.
8. Using the same 25-ml pipet, fill the rest of the gel with the 0.5× TBE/gel solution.
9. Insert combs, position clamps, and observe polymerization as in steps 9 and 10 of
basic protocol.
10. Run and process the gel as in steps 11 to 38 of basic protocol.
ELECTROLYTE-GRADIENT SEQUENCING GELS
An alternative way to generate an ionic-strength gradient in a gel has been developed by Sheen
and Seed (1988). In this protocol, the ionic strength of the bottom of the gel is increased
simply by increasing the salt concentration in the bottom buffer chamber. During the run, the
salt is electrophoresed into the gel and generates a reproducible and effective gradient.
ALTERNATE
PROTOCOL
Additional Materials
0.5× and 1× TBE buffer (APPENDIX 2)
3 M sodium acetate unbuffered
1. Pour and prerun the sequencing gel as described in steps 1 to 19 of basic protocol.
Use 0.5× TBE buffer in the top and 1× TBE buffer in the bottom reservoir.
2. Prepare and load sequencing samples as in steps 20 to 22 of basic protocol.
3a. If ≤400 bases of sequence information is needed, add 3 M sodium acetate to the
bottom reservoir to a final concentration of 1 M. Proceed to step 4.
3b. If ≥400 bases of sequence information is desired, proceed to step 4 and wait 2 to 3
hr after beginning the electrophoresis to add 3 M sodium acetate to the bottom
reservoir to a final concentration of 1 M.
4. Run gels at 60 W constant power.
It will take about 75% longer than usual for the dye markers to migrate to the same location
on the gel.
The temperature of the gel should be monitored carefully. If the gel becomes significantly
hotter at the top than the bottom, reduce the power.
5. Process the gel as described in steps 25 to 38 of basic protocol.
DNA Sequencing
7.6.8
Current Protocols in Molecular Biology
Supplement 16
ALTERNATE
PROTOCOL
FORMAMIDE-CONTAINING SEQUENCING GELS
In this protocol, formamide is added to the acrylamide gel solution (U.S. Biochemical,
1990) to destabilize the secondary structures of sequencing products that can cause the
anomalous migration of bands known as compressions (UNIT 7.4).
Additional Materials
Formamide gel solution
5% acetic acid/20% methanol (v/v) fixer solution
1. Assemble gel sandwich as in steps 1 to 4 of basic protocol.
2. Prepare formamide gel solution according to the acrylamide concentration desired.
Heat gently with stirring until dissolved. Cool solution until it is ≤30°C.
Do not let the temperature of gel solution exceed 55°C.
Filter solution through Whatman No. 1 filter paper if particulate matter is visible.
3. Add 0.15 ml TEMED and 1 ml of 10% ammonium persulfate. Immediately pour the
solution into the gel sandwich. Observe polymerization.
The gel solution is viscous. Hold plates at a nearly vertical angle while pouring the
solution—this is easiest if the solution is in a beaker.
The gel should polymerize within 30 min.
4. Run the gel 45 to 70 W, constant power.
This requires a higher voltage (60% higher) than nonformamide-containing gels. The DNA
migrates about half as fast.
5. Process and dry the gel as described in steps 25 to 38 of basic protocol, except fix the
gel 15 min in 5% acetic acid/20% methanol fixer solution in step 31a.
20% methanol prevents the gel from swelling during the fixing step.
REAGENTS AND SOLUTIONS
38% acrylamide/2% bisacrylamide
Mix 38 g of acrylamide (ultrapure) and 2 g N,N′-bismethylene acrylamide (ultrapure) in ∼60 ml H2O. Heat if necessary to dissolve, but do not heat above 55°C.
Adjust volume to 100 ml. Store in a brown jar for up to several months at 4°C.
Discard if an ammonia smell is detected.
CAUTION: Acrylamide is a neurotoxin. Handle with gloves.
Ultrapure acrylamide and bisacrylamide are available from numerous commercial sources.
Many of these suppliers also offer premixed acrylamide/bisacrylamide in both solid and
liquid form.
Deionization and filtering are usually not needed when using ultrapure reagents. If deionization is desired, add 5 g Amberlite MB-1 (Sigma) or equivalent mixed bed resin and stir 30
min at room temperature. Filter through Whatman No. 1 paper.
Denaturing Gel
Electrophoresis
for Sequencing
7.6.9
Supplement 16
Current Protocols in Molecular Biology
Buffer-gradient gel solutions
0.5× TBE (clear solution):
Acrylamide concentration (%)
Reagent
Urea (ultrapure) (g)
38% acrylamide/2% bisacrylamide (ml)
10× TBE (APPENDIX 2) (ml)
H2O (ml)
Total volume (ml)
4
6
8
21
5.0
2.5
26.5
50.0
21
7.5
2.5
24
50.0
21
10.0
2.5
21.5
50.0
2.5× TBE (blue solution):
Reagent
Urea (ultrapure) (g)
38% acrylamide/2% bisacrylamide (ml)
10× TBE (APPENDIX 2) (ml)
Sucrose (g)
1% (wt/vol) bromphenol blue (µl)
H2O (ml)
Total volume (ml)
Acrylamide concentration (%)
4
6
8
10.5
2.5
6.25
2.5
250
6.25
25.0
10.5
3.75
6.25
2.5
250
5.0
25.0
10.5
5.0
6.25
2.5
250
3.75
25.0
Denaturing acrylamide gel solution
Reagent
Urea (ultrapure) (g)
38% acrylamide/2% bisacrylamide (ml)
10× TBE (APPENDIX 2) (ml)
H2O (ml)
Total volume (ml)
Acrylamide concentration (%)
4
6
8
25.2
6.0
6.0
27
60
25.2
9.0
6.0
24
60
25.2
12.0
6.0
21
60
Filter solution through Whatman No. 1 filter paper. Quantities are for a single sequencing
gel. If gels are poured daily, make gel solutions in quantity—e.g., make 1 liter of gel solution
by multiplying the above quantities by 16.7. Store 2 to 4 weeks at 4°C. Solutions of
acrylamide deteriorate quickly, especially when exposed to light or left at room temperature.
Formamide gel solution
Acrylamide concentration (%)
Reagenta
Urea (ultrapure) (g)
38% acrylamide/2% bisacrylamide (ml)
10× TBE (APPENDIX 2) (ml)
Deionized formamide (ml)b
H2O (ml)
Total volume (ml)
4
6
8
42
10
10
40
10
100
42
15
10
40
5
100
42
20
10
40
0
100
aHeat
the ingredients in a beaker while stirring. When dissolved, add H2O to 100 ml total volume.
formamide by stirring with Amberlite MB-1 (Sigma) or equivalent mixed-bed resin 30 min
at 4°C. Filter through Whatman No. 1 filter paper and store at −20°C. Formamide that has been deionized
is available from American Bioanalytical.
bDeionize
DNA Sequencing
7.6.10
Current Protocols in Molecular Biology
Supplement 16
COMMENTARY
Background Information
Refer to the Chapter 2 introduction for a
general discussion of gel electrophoresis of
nucleic acids and gels as electric circuits.
Critical Parameters and
Troubleshooting
Silanizing the plates
After electrophoresis, the gel is supported
by one of the glass plates during processing. As
the gel sandwich is disassembled, however, the
gel often sticks to both plates because polymerized acrylamide is inherently sticky, especially after being heated during electrophoresis.
Silanization helps to prevent this common
problem by aiding the release of the gel from
one of the plates when the gel sandwich is taken
apart; annotations to the steps describing processing of the sequencing gel offer suggestions
for coping when the problem does occur. The
basic protocol recommends silanizing both gel
plates; an alternative strategy is to silanize one
plate (usually the short, notched plate) to increase the chance that gel will stick to untreated
plate.
An alternate silanizing agent is Sigmacote
(Sigma #SL-2), which requires overnight drying or drying at 90°C (see manufacturer’s instructions). The advantage of Sigmacote is that
it is relatively permanent and only has to be
reapplied when the gel starts sticking to the
glass plates during subsequent processing. The
untreated side of each plate should be marked
with a piece of tape to make sure that the
silanized side always faces the gel.
Denaturing Gel
Electrophoresis
for Sequencing
Choosing acrylamide gel concentrations
As the percentage of acrylamide in the gel
decreases, the pore size increases, allowing
resolution of larger oligonucleotides. Thus,
lower acrylamide percentages (4% to 6%)
allow more sequence to be read from a single
gel than does an 8% gel. However, these gels
are harder to handle due to increased fragility
and softer texture, and the banding pattern
may not be quite as sharp as those observed
in 8% gels.
We recommend running 6% acrylamide,
nongradient gels as a starting point for the
novice. Two gels can be run, one for a relatively
short time to retain the shorter oligonucleotides, the second for a longer time to maximize
separation of the longer oligonucleotides. This
two gel procedure, using 6% acrylamide, al-
lows 300 to 350 bases of sequence information
to be obtained from a single set of sequencing
reactions. If fewer than five sets of sequencing
reactions have been performed, one 6% gel can
be used for both the short and the long runs (the
typical gel format has room for ten sets of
sequencing reactions, or 40 samples). An alternative practice is to use an 8% gel for the shorter
oligonucleotides and a 6% gel for the longer
oligonucleotides (see below for a discussion of
electrophoresis times for each of the above
examples). As experience is gained with manipulation of gels, modifications of the basic
protocol, as described in the introduction, can
be incorporated to increase the amount of sequence information gained from each set of
reactions.
Precipitating TBE
10× TBE buffer at pH 8.3 to 8.6 (as described
in APPENDIX 2) tends to precipitate during longterm storage. Adjusting the pH to 8.9 with
NaOH prevents this precipitation without affecting the resolution of the sequencing gel
(Mayeda and Krainer, 1991). The TBE buffer
in the gel and buffer reservoirs should be prepared from the same 10× stock.
Leaking combs
Sharkstooth combs have a smooth edge that
forms the top of the gel during polymerization
and a “sharkstooth” edge containing teeth that
form the sample wells when the points of the
teeth are inserted into the top of the gel after
polymerization. Lanes created by sharkstooth
combs are spaced more closely together than
those made with a preformed-well comb, thus
allowing more samples to be loaded on a gel
and yielding a sequencing ladder that is easier
to read. Care must be taken to preserve the
points of the teeth since they will make the
wells. Even when all the teeth are intact, sharkstooth combs have a tendency to leak between
the wells because variations in the comb’s thickness can result in a space between the teeth and
the glass plates when the combs are reinserted to
form wells. Leakage can be tested before loading samples by applying a small amount of
loading dye into each well and observing whether
the dye flows into adjacent lanes. Those wells
that leak can then be omitted from use. This test
dye should be run into the gel a short distance
before applying the actual samples so that
loading can be tracked. Alternatively, for
minor leaking, a set of four reactions can be
7.6.11
Supplement 16
Current Protocols in Molecular Biology
loaded and run into the gel before the next set
is applied. Another trick is to apply a tiny
amount of Vaseline on the points of the teeth
before reinserting them into the gel. This seals
the bottoms of the wells.
“Smiling” gels
Level, “nonsmiling” (nondistorted) bands
require even heat dissipation across the gel. To
achieve this, both buffer surfaces and the bottom and top of the gel must be aligned parallel
to each other. The alignment of the bottom
buffer can be a particular problem because it
tends to climb up between the back glass plate
and the electrophoresis apparatus due to capillary action when the gel sandwich is clamped
to it. This can be prevented by inserting a small
piece of folded filter paper between the gel
sandwich and the electrophoresis apparatus, ∼5
cm above the level of the bottom buffer. This
forces the bottom of the sandwich away from
the apparatus, allowing the bottom buffer to
form a level surface.
In addition, deformation of the gel in the
apparatus can contribute to uneven heat dissipation. If the gel sandwich is assembled with
tape rather than clamps, distortion can be prevented during polymerization by clamping the
gel plates with book-binder clamps at the same
positions in which they will attach the gel
sandwich to the electrophoresis apparatus. This
maintains the same pressure points on the gel
when it is polymerizing. Some commercially
available gel setups allow the gel to be poured
in the gel apparatus, avoiding this complication.
To ensure even conduction of the heat generated during electrophoresis, an aluminum
plate (0.4 cm thick, 34 × 22 cm) can be clamped
onto the front glass plate with the same bookbinder clamps used to hold the gel sandwich to
the apparatus. The aluminum plate must be
positioned so that it does not touch any buffer
during electrophoresis. Some commercially
available gel setups have this feature, or have a
top buffer reservoir that extends down the rear
of the apparatus to evenly distribute heat over
the gel.
Reading beyond 350 nucleotides
To unambiguously read DNA sequence beyond 350 nucleotides, it is essential to prevent
distorted bands as described above. In addition,
a concentration of ≥5% glycerol in the gel
samples results in distortion of bands in the
450- to 550-nucleotide range (Tabor and
Richardson, 1987); this can be avoided by diluting the sequencing polymerase in diluent
without glycerol (UNIT 7.4). The modifications to
the basic protocol resulting in various types of
gradient gels (see background information)
will generally increase the ability to read the
sequence in the 350-nucleotide range. A rather
infrequently used tactic is to load duplicate sets
of each reaction in the order GATCGTAC; this
facilitates correct ordering of bands ≥350 nucleotides because each reaction is run on the
gel next to each of the other three reactions.
Electrophoresis times and coordinating
multiple gels
To decide how long to run the gel that will
retain the shortest oligonucleotides (“short”
gel), the length of the primer and how far away
from the beginning of the target DNA synthesis
will be initiated must be known. Add those two
numbers together and use Table 7.6.2 as a
guideline for bromphenol blue dye migration.
Adjust the electrophoresis time so that some
polylinker sequences can be retained on the gel
and identified. The “long” gel should be run so
that the shortest oligonucleotides remaining on
this gel overlap by at least ten bases with the
longest oligonucleotides on the short gel.
Experience is the best guide in the coordination of the gels. The following examples may
serve as initial references.
If two 6% gels and the −40 sequencing
primer (a 24-mer that initiates DNA synthesis
40 nucleotides from the M13 polylinker; New
England Biolabs) are used, run the first gel 10
to 15 min after the bromphenol blue runs off
the bottom of the gel. Run the second gel until
the xylene cyanol is almost at the bottom of the
gel (∼5 hr).
If an 8% gel is used for the shorter oligonucleotides and a 6% gel for the longer oligonucleotides, run the 8% gel until the bromphenol
blue runs off and the xylene cyanol has run
through ∼40% of the gel (∼2 hr). Run the 6%
gel until the xylene cyanol is at the bottom of
the gel (∼5 hr).
If the same 6% gel is used for both the short
and long runs, load one set of reactions and run
the gel until the bromphenol blue is at the
bottom. Turn off the power to the gel and load
a duplicate set of reactions on the unused part
of the gel. Continue running the gel until the
duplicate bromphenol blue is at the bottom of
the gel. The xylene cyanol dye from the first set
of reactions should be at the bottom of the first
(long) run.
DNA Sequencing
7.6.12
Current Protocols in Molecular Biology
Supplement 16
Time Considerations
Pouring, running, and processing of a sequencing gel is usually interspersed with performing sequencing reactions (UNIT 7.4). The
typical gel format has room for ten sets of
reactions generating 40 samples. Larger gel
formats (up to 78 lanes) are available commercially but we recommend that the novice run
and process no more than two 40-sample format gels at one time. This can be accomplished
in a single long day, typically 7 to 8 hr for two
gels. An experienced sequencer can typically
perform the process for four gels in 9 to 10 hr.
Sequencing gels can be prepared up to 48 hr in
advance of use; assembling and pouring the gel
takes ∼1 hr.
Literature Cited
Ansorge, W. and Labeit, S. 1984. Field gradients
improve resolution in DNA sequencing gels. J.
Biochem. Biophys. Methods 10:237-243.
Biggin, M.D., Gibson, T.J., and Hong, G.F. 1983.
Buffer gradient gels and 35S-label as an aid to
rapid DNA sequence determination. Proc. Natl.
Acad. Sci. U.S.A. 80:3963-3965.
Brown, N.L. 1984. DNA sequencing. Methods Microbiol. 17:259-13.
DNA sequencing gels by the use of a fieldstrength gradient. J. Biochem. Biophys. Methods
10:83-90.
Sheen, J.-Y. and Seed, B. 1988. Electrolyte gradient
gels for DNA sequencing. BioTechniques 6:942944.
Slatko, B. 1991a. Sources of reagents and supplies
for dideoxy DNA sequencing and other applications. In Methods in Nucleic Acids Research (J.
Karam, L. Chao, and G. Warr, eds.) pp. 379-392.
CRC Press, Boca Raton, Fla.
Tabor, S. and Richardson, C.C. 1987. DNA sequence analysis with a modified bacteriophage
T7 DNA polymerase. Proc. Natl. Acad. Sci.
U.S.A. 84:4767-4771.
U.S. Biochemical. 1990. Formamide gels (40%) for
sequencing DNA. Comments 17(1):31.
Williams S.A., Slatko, B.E., Moran, L.S., and DiSimone, S.M. 1986. Sequencing in the fast lane: A
rapid protocol for [α-35S]dATP dideoxy DNA
sequencing. BioTechniques 4:138-147.
Key Reference
Slatko, B. 1991b. Protocols for manual dideoxy
DNA sequencing. In Methods in Nucleic Acids
Research (J. Karam, L. Chao, and G. Warr, eds.)
pp. 83-129. CRC Press, Boca Raton, Fla.
Contains references for numerous modifications to
the basic sequencing gel protocol.
Isfort, R. and Ihle, J. 1988. The 4-6-8 method of
sequence analysis. BioTechniques 6:138-141.
Mayeda, A. and Krainer, A.R. 1991. Long-term
storage of concentrated Tris borate-EDTA electrophoresis buffers without precipitation.
BioTechniques 10:182.
Contributed by Barton E. Slatko
New England Biolabs
Beverly, Massachusetts
Olsson, A., Moks, T. Muhlen, J., and Gaal, A.B.
1984. Uniformly spaced banding patterns in
Lisa M. Albright
Reading, Massachusetts
Denaturing Gel
Electrophoresis
for Sequencing
7.6.13
Supplement 16
Current Protocols in Molecular Biology