Protocol 4 Electrophoresis

1. Carefully remove the comb and insert the cassette into the gel apparatus according to the manufacturer's instructions.

2. Add sufficient volume of 0.6X TBE running buffer (diluted from 5X TBE stock buffer) to top (cathode) and bottom (anode) chambers. Gently wash the top surface of the gel three times by pipetting the running buffer up and down.

3. Vertically hold the left and right sides of the comb using two hands and carefully insert the comb into the top of the sandwich to form the wells. The insertion of the comb should be done slowly and evenly until all the teeth just touch the surface of the gel. Remove any air bubbles trapped in the wells using a pipette.

Tips: It is acceptable if the teeth of the comb penetrate a little bit into the gel (<0.5 mm from the surface of the gel). However, if the teeth are inserted too deeply into the gel or the comb repeatedly pulled and inserted, the surface of the gel will be badly damaged and cause leaking when loading samples. This results in contamination of the wells and inaccurate sequence data.

4. Calculate the volume of the gel (the length x the width x the thickness of the spacer) and set the power supply. We strongly recommend setting it at a constant power (watts) using 1.0 to 1.1 W/cm3. If constant power is not available from the power supply, 0.5 to 0.8 mA/cm3 constant current should be set up. For example:

Gel volume Constant watts Electrophoresis temperature

35 cm3 35 to 38.5 45 to 550C

70 cm3 70 to 77 45 to 550C

Caution: If the power supply is set at a nonconstant level (e.g., high voltage or high current), the gel may burn or melt during the electrophoresis due to the amount of heat generated.

5. Connect the power supply unit to the gel apparatus with the cathode at the top of the gel and anode at the bottom of the gel so that the negatively charged DNA will migrate downwards.

6. Turn on the power and prerun the gel for 10 to 12 min.

7. After prerunning for 7 to 9 min, denature the labeled DNA samples at 75°C for 2.5 to 3 min prior to loading in wells. The tubes containing the samples should be capped tightly while heating in order to prevent evaporation of the samples.

Tip: For heating, placing the tubes in a heating block in the order of A, T, G, C or A, G, T, C is recommended to in order to prevent a potential mix-up when loading the samples into the gel.

8. When pre-electrophoresis is complete, turn off the power supply unit, wash the wells briefly to remove urea, and then quickly and carefully load the samples into appropriate wells (3.5 to 4 ml/well).

Tips: (a) No leaking is allowed between wells. Otherwise, the DNA samples will be contaminated and result in a mix-up of the nucleotide sequences. (b) Special pipette tips are available for DNA sample loading. In order to prevent any contamination, using one tip for loading multiple samples is not recommended . If a 0.4-mm spacer is used, then using normal 100- to 200-fl pipette tips works fine for loading. This is faster and no air bubbles develop, which is quite common when using the commercial sequencing loading tips with a long and flat tip. (c) After taking a sample from each tube, the tubes should be capped immediately. All tubes should be briefly spun down and stored at 4 or -20°C until the next loading. (d) It is very important that the samples loaded into the wells be recorded in a notebook in the order of the DNA samples loaded (e.g., A, G, T, C or A, T, G, C depending on particular loading sequence). This will help in reading the DNA sequence following autoradiography.

9. After all the samples are loaded, turn on the power supply and allow electrophoresis to occur at an appropriate constant voltage. The running time depends on the size of the DNA. Generally, the run times for 250 bp, 400 to 450 bp and > 500 bp are 2, 4, and 6 to 8 h, respectively. For multiple-loading (e.g., two to five times loading depending on the size of the gel and volume of the DNA sample), monitor the migration of the first blue dye (Bromophenol blue or BPB) that migrates at 40 bp. When the BPB reaches approximately 2 to 3 cm from the bottom, turn off the power supply and carry out the next loading so that there are overlapping sequences between the adjacent loads. This is very useful for correctly reading DNA sequences after autoradiography.

Tips: The power supply must be turned off and the wells for sample loading should be washed to remove air bubbles with the running buffer using a pipette. DNA samples may be denatured again at 75°C for 2 min prior to loading. The loading order of the samples should be the same as for the previous loading to avoid misreading DNA sequences.

10. When the electrophoresis is complete, disconnect the power supply and decant the upper chamber buffer. Lay the cassette containing the gel on a table or bench and allow it to cool for 20 to 25 min prior to separating the glass plates. Mark and measure the area of the gel to be transferred.

Note: If the glass plates are separated immediately following electrophoresis, the gel may not be flat, may become broken or cause difficulty in separating the glass plates.

11. While the gel is cooling, wear clean gloves and cut one piece of nylon membrane and three pieces of 3MM Whatman paper. The size of membrane or filter paper should be slightly larger than the area of the gel to be transferred. Using a pencil, label one corner of the membrane for orientation and prewet the membrane in dd.H2O for 5 to 10 min.

12. Remove the clamps from the glass plates. Starting at one corner of the spacer site, carefully insert a spatula into the sandwich and slowly lift the top glass plate (the Sigma-coated plate) with one hand. Meanwhile, hold the bottom plate with the other hand until the top glass plate is completely separated from the gel. The gel should remain stuck to the bottom plate.

Tip: Separation of glass plates is critical for the success of DNA sequencing. As long as the top plate is loosened, continue to lift it until it is completely separated from the other plate. Do not allow the top plate to come back in contact with the gel at any time during separation or it may cause trouble for the gel.

13. Perform fixation. If Long Ranger Gel mixture is used, the electrophoresed gel does not need to be fixed. However, if a regular acrylamide gel mixture is used, the gel should be fixed. Immerse the gel together with the bottom plate in a relatively large tray filled with a sufficient volume of fixing solution containing 15 to 20% of ethanol or methanol and 5 to 10% acetic acid for 15 to 20 min. Remove the gel together with the plate from the tray, drain off excess fixing solution and gently place bleached, clean paper towels on the gel to remove excess solution.


1. Transfer the sequenced DNA from the gel onto the prewetted membrane in the order shown: wear clean gloves and lay the membrane on the area of the gel to be transferred with the marked corner facing the gel. Remove any air bubbles underneath the membrane by gently rolling a clean 25ml pipette on the membrane. Carefully place three layers, one by one, of 3MM Whatman dry filter papers on the membrane. Place the other glass plate or its equivalent on top of the filter papers and then a weight on the top (e.g., a bottle or beaker containing 1000 ml water). Leave undisturbed for 1 to 1.5 h.

Note: Any bubbles generated will block local transfer of DNA bands from the gel to the membrane. Once this occurs, reading of DNA sequences will not be accurate. One virtually must start all over again.

2. Remove the weight, top glass plate and 3MM filters. Wear gloves and gently peel the membrane off the gel and place the membrane with the DNA side facing up on a sheet of clean 3MM Whatman paper. Allow the membrane to dry for 30 to 45 min at room temperature. The dried membrane may be stored at 4°C until use.


1. Block nonspecific binding sites on the membrane with 0.4 ml/cm2 of blocking solution using an appropriate size of tray. Place the tray on an orbital shaker with shaking at 60 rpm for 40 min.

2. Add streptavidin-alkaline phosphatase conjugate (SAAP) to the blocking solution (1:2000 to 5000 dilution) in the tray. Allow SAAP to hybridize with biotinylated primer or nucleotides on the membrane for 40 min with shaking at 60 rpm.

3. Briefly rinse the membrane with dd.H2O and wash it with 1 ml/ cm2 of 0.5 to 1.0X SSC containing 0.1% SDS by agitating for 10 min on a shaker at 60 rpm. Repeat this step twice.

4. Briefly rinse the membrane with dd.H2O and wash it once with 1 ml/ cm2 of 0.5 to 1.0X SSC without SDS for 5 min on a shaker at 60 rpm. The membrane can be subjected to detection by the following methods.

Method A. Chemiluminescent Detection

1. Place a piece of SaranWrap film on a bench. According to the size of the membrane, add 10 ml/cm2 of Lumi-Phos 530 (Boehringer Mannheim Corporation) to the center of the SaranWrap.

2. Wear gloves and briefly dampen the membrane to remove excess washing solution and thoroughly wet the DNA-binding side of the membrane by lifting and overlaying the membrane several times.

3. Wrap the membrane with SaranWrap film, leaving two ends of the film unfolded. Place the wrapped membrane on a paper towel and carefully press the wrapped membrane, using another piece of paper towel, to wipe off excess detection solution through the unfolded ends of the SaranWrap. Excess detection solution will most likely cause a high background.

4. Completely wrap the membrane and place it in an exposure cassette with the DNA side facing up. Tape the four corners of the wrapped membrane.

5. In a darkroom with the safe light on, overlay the membrane with a sheet of Kodak® XAR or XRP x-ray film and close the cassette. Allow exposure to proceed at room temperature for 20 s to 12 h, depending on the intensity of the detected signal.

6. In a darkroom, develop and fix the film in an appropriate developer and fixer, respectively. If an x-ray processor is available, development, fixation, washing and drying of the film can be completed in 2 min.

Tips: Multiple films may be needed to be exposed and processed until the desired signals are obtained. Exposure for more than 4 h may generate a high black background. In our experience, a good hybridization and detection should display sharp bands within 1.5 h (Figure 5.1).

7. Proceed to DNA sequence reading (Figure 5.2).

Method B. Colorimetric Detection

1. Following washing, place the membrane in color developing solution (20 ml/cm2) containing 0.08 ml of NBT stock solution and 0.06 ml of BCIP stock solution. NBT and BCIP stock solutions are commercially available.

2. Allow color to develop in a dark place for 15 to 120 min at room temperature or until a desired level of detection is obtained. Detected bands should appear as a blue-purple color on the membrane.

Reagents Needed

5X TBE Buffer

600 ml dd.H2O

Dissolve well after each addition. Add dd.H2O to 1 l. Autoclave.

20X SSC Solution (1 l) 175.3 g NaCl 88.4 Sodium citrate Adjust the pH to 7.5 with HCl.

Blocking Buffer

3% (w/v) Nucleic acid blocking reagents or 5% (w/v) nonfat dry milk

150 mM NaCl

Dissolve well with stirring.

Color Developing Buffer 0.1 M Tris-HCl, pH 9.5 0.1 M NaCl 50 mM MgCl2

NBT Stock Solution

75 mg/ml Nitroblue tetrazolium (NBT) salt in 70% (v/v) dimethylformamide

FIGURE 5.1 Chemiluminescent detection of DNA sequences.

FIGURE 5.2 Reading of DNA sequences.

BCIP Stock Solution

50 mg/ml 5-Bromo-4-chloro-3-indolyl phosphate (BCIP or X-phosphate), in 100% dimethylformamide

Streptavidin-Alkaline Phosphatase Conjugate (SAAP)

Lumi-Phos 530


Procedures for isotopic DNA sequencing are essentially the same as those described for the nonisotopic sequencing method except for the following differences.

The first difference is the labeling reaction in which [35S]dATP or [35S]dCTP (not a biotinylated nucleotide) is employed. Add the following components to the annealed primer-template mixture (10 ml).

Diluted labeling mixture, 2 ml

[35S]dATP or [35S]dCTP (1000 to 1500 Ci/mmol), 1 ml

Diluted Sequenase DNA polymerase, 2 ml

The second difference is in the drying of the gel and autoradiography. No DNA transfer is involved. Specific steps are:

1. Once the top glass plate is separated from the gel on the bottom plate, gently and slowly lay the precut 3MM Whatman paper on the gel from the bottom side or the upper side of the gel until it lies on the entire gel. Gently and firmly press the paper thoroughly onto the gel surface with a styrofoam block (approximately 50 cm long, 5 cm wide and 5 cm thick). Then, starting from one corner, slowly peel the 3MM Whatman paper with the gel on it from the bottom plate. Place it on a flat surface with the gel side up and carefully cover the gel with SaranWrap without any air bubbles or wrinkles generated between the SaranWrap and the gel.

2. Assemble the gel on the drying apparatus according to the manufacturer's instructions and dry the gel at 70 to 80°C under vacuum for 30 to 55 min, depending on the strength of the vacuum pump. Turn off the heat and allow cooling for another 20 to 30 min under vacuum. Proceed to auto-radiography.

Tips: (a) Place a water trap at a position lower than the gel dryer for effective suction of the water. If no freeze or refrigerated trap is available, place a water-trap flask or bottle in a bucket with some dry ice and ethanol. Connect the trap to a vacuum pump and to the gel dryer. This works fine in our laboratory. (b) If the gel is removed immediately after the heat is turned off, it may be cracked or have an uneven surface. Cooling the gel for a while under vacuum before taking it out of the gel dryer is recommended.

3. Peel the SaranWrap from the gel and put the gel on an appropriate exposure cassette. In a darkroom under a safe light, place an appropriately sized piece of x-ray film (Kodak XAR-film or Amersham x-ray film) on the surface of the gel and close the cassette. Allow exposure to take place at room temperature (for 35S) by placing the cassette in a dark place for 1 to 4 days, depending on the intensity of the signal. It is recommended that the film be developed after exposure for 24 h and then, if necessary, the length of further exposure decided.

Caution: [35S] dATP or dCTP is dangerous. Care should be taken when using the nucleotides. Gloves should be worn and an appropriate Plexiglas protector should be used. Any used isotopic buffer should be collected in a special container for disposal.

Compression is a common phenomenon that usually causes unreadability on a developed sequencing film. The problem is primarily due to intramolecular base pairing or G-C rich in a primer extension. The local folded structures or hairpin loops migrate faster through the gel matrix than unfolded structures, and persist during the electrophoresis. As a result, bands run very close together with a gap or increased band spacing in the region. One way to solve this problem is to increase the denaturing conditions in the gel matrix using urea and formamide. The procedures for preparing a formamide gel, electrophoresis and autoradiog-raphy are very similar to those described in earlier protocols except for the following steps.

1. The gel mixture:

Use of Long Ranger Gel Mixture from AT Biochemicals

Components Small size gel (50 ml) Large size gel (100 ml)

Ultrapure urea

21 g

42 g

Long Ranger mixture

8 ml

16 ml

Ultrapure formamide

20 ml

40 ml


10 ml

20 ml

Add dd.H2O up to

50 ml

100 ml

Use of Regular Acrylamide Gel Mixture if Sequenase Version 2.0 DNA Polymerase is Diluted with a Buffer without Glycerol



Small size gel (50 ml) Large size gel (100 ml)

Ultrapure urea (7 M) Ultrapure formamide 6% Acrylamide/fe-acrylamide 8% Acrylamide/fe-acrylamide 5X TBE

Add dd.H2O up to

21 g 15 to 20 ml 2.8 g/0.15 g 3.8 g/0.2 g 5 ml 50 ml

42 g

Recipe for a Regular Acrylamide Gel Mixture if Sequenase Version 2.0 DNA Polymerase is Diluted in Glycerol Enzyme Dilution Buffer


Ultrapure urea (7 M) Ultrapure formamide 6% Acrylamide/fe-acrylamide 8% Acrylamide/bis-acrylamide 10X Glycerol tolerant gel buffer Add dd.H2O up to

Small size gel (50 ml)

21 g 15 to 20 ml 2.8 g/0.15 g 3.8 g/0.2 g 5 ml 50 ml

Large size gel (100 ml)

42 g 30 to 40 ml 5.7 g/0.3 g 7.6 g/0.4 g 10 ml 100 ml

2. Electrophoresis: double the electrophoresis time because formamide slows the migration of DNA by about 50%.

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