Submarine agarose gel electrophoresis

Agarose gels are the preferred method of separating and purifying DNA fragments of 100-10,000 bp in length. For routine work, 6.5 x 9.0 x 1.0 cm minigels with 8-well combs which hold 20 μL samples per well are convenient. The exact method of pouring a gel will depend on the agarose gel apparatus used, but some general techniques are applicable to nearly all apparatus:

• Prepare agarose gel stock solutions by mixing agarose or low-melt agarose¹ with 1x TAE buffer. This is conveniently done in a 500 mL Wheaton bottle. For a 1% gel, add 1 g of agarose for every 100 mL of TAE buffer. Typically no more than 200 mL are made at one time.²
• Loosen the cap and microwave agarose for approx. 1 min for every 100 mL of agarose solution. Swirl to mix thoroughly but without introducing bubbles. The agarose should be fully melted before proceeding. If necessary microwave for additional time to fully melt agarose.
• If the solution is being used for the first time, add 50 μL of ethidium bromide solution (10 mg/mL) to the agarose solution.
• Allow the agarose solution to cool enough so that it can be held in the bare hand, and pour the gel and introduce the comb. Allow the gel to harden. Once the gel takes a partial set, it can be placed in a –20 °C freezer for 5-10 minutes to speed complete hardening.
• Gently remove the comb,³ place the gel in the apparatus, and fill the apparatus with 1x TAE buffer deep enough to cover the gel completely 2-3 mm deep.
• Load samples with a pipettor by placing the tip just below the top of the well. The high density of the loading buffer will ensure the samples sink to the bottom of the well.
• Verify that the electrode leads are attached so that the negative (black) electrode is closest to the loading wells. DNA will migrate from the negative (black) electrode to the positive (red) electrode. Also verify that the red and black leads are attached to the correct sources on the power supply. (Red to red, black to black.)
• Run the samples out at 120 V constant⁴ for approximately 30-40 min as desired to achieve the necessary separation.
• Disconnect the power, remove the gel tray, and place it on a UV transilluminator. Don a UV face shield or UV-absorbing glasses or goggles before engaging the UV lamp. DNA will appear as orange bands within the gel.
• Gels are conveniently photodocumented with an ordinary digital camera equipped with a dark yellow (B&W #023) filter.

Notes

1. Agarose is much cheaper than low-melt agarose. The latter is used for preparative purposes, when bands are sliced out of the gel and the DNA must be recovered for further use. The former can be used for analytical purposes.
2. Additional agarose and TAE buffer can be added directly to partially used bottles of agarose as needed; it is not necessary to remove remaining agarose and buffer prior to making additional solution.
3. Rips and tears in the wells can be minimized by pouring a little 1x TAE buffer on the top of the gel around the comb to lubricate its removal.
4. The literature usually recommends much lower voltages for agarose gel separations, with consequently longer separation times. While lower voltages result in better resolution, it is not necessary for routine recombinant DNA work.

Recovering DNA fragments from agarose gels or solution

One of the most convenient methods for recovering purified DNA bands visualized in ethidium bromide stained agarose gels involves cutting out gel slices and purifying/recovering on silica gel using a commercial product (Geneclean Turbo, Zymoclean, etc.)

• Select the desired DNA band and, using a knife,¹ make vertical slices in front of, behind, and to each side of the band to free it from the rest of the gel.
• Slipping the knife into the cut previously made below the gel band, gently tip the knife and lift the gel slice from the rest of the gel, and place in a tared microcentrifuge tube. A typical gel slice should weigh 100-250 mg.
• If necessary, slices may be frozen at this stage for later processing.

Purification using Gene-Clean Turbo

• To each gel slice, add an equal volume of Gene-Clean Turbo salt solution and mix well²
• Heat at 50-55 °C for 5 min or until gel slice has completely melted
• Mix solution well and transfer (<600 μL ) to a Geneclean Turbo cartridge
• Place cartridge into a catch tube and spin at 6,000 rpm for 1 min, then pulse at 14,000 rpm; empty catch tube as needed. The cartridge should be free of liquid before proceeding.
• Add 500 μL of Geneclean Turbo Wash solution to the cartridge; spin at 6,000 rpm for 1 min, then pulse at 14,000 rpm; empty the catch tube as needed.
• Repeat the previous wash step
• Empty catch tube and spin at 14,000 rpm for 2 min to completely remove wash solution from the spin filter
• Transfer the cartridge to a clean 1.5 mL microcentrifuge tube
• Add 30 μL of water to the cartridge filter and allow to stand for 5 min
• Spin at 14,000 rpm for 2 min to collect the DNA
• For most uses, the DNA solution should be evaporated to dryness in a vacuum evaporator, and stored at –20 ºC until needed.
• To reconstitute DNA solution, add 10 μL of water to the bottom of the microcentrifuge tube, flick or vortex gently to mix, and centrifuge briefly to collect solution at the bottom of the tube. Use immediately, or store at –20 ºC.

Purification using Zymoclean Gel DNA Extraction Kit

This method is currently the preferred method for gel product extraction and purification.

• Tare an empty 1.5 mL eppendorf tube and weigh the samples.
• Take the heaviest sample weight and multiply by 3 to determine the amount of ADB to add to all samples (e.g. for 100 μL (mg) of agarose gel slice add 300 μL of ADB).
• Incubate at 55 ºC for 5 minutes until the gel slice is completely dissolved.
• Mix solution well and transfer (max 500 μL) to a Zymo-Spin I Column in a Collection Tube. If more than 500 μL are to be processed, spin sample in batches of 500 μL or less.
• Centrifuge at 13,000 x g for 30 seconds. Discard the flow-through.
• Add 200 μL of Wash Buffer to column and centrifuge at 13,000 x g for 30 seconds. Discard the flow-through. Repeat the wash step.
• Add 10 μL of water directly to the column matrix and wait 1 minute. Place column into a 1.5 mL tube and centrifuge at 13,000 x g for 30 seconds to elute DNA.

Notes

1. A satisfactory knife can be constructed by taking a standard thin, flat spatula, grinding each end flat, and sharpening, if necessary. Such a knife is about the same width as a gel lane in an 8-well agarose gel apparatus.
2. If purifying DNA from solution, e.g., restriction digests of PCR products, add at least 5x volume of GeneClean Turbo solution and mix well. Omit the heating step and then follow the GeneClean Turbo protocol othwerwise as described. Alternatively, for Zymoclean, add at least 3x volume of ADB solution to the digestion product (50 μL works well for a 15 μL digest solution).

SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

Preparing gels

For routine work, precast 10 cm x 10 cm x 1 mm minigels are convenient. However, the following protocol is appropriate for preparing four (4) homemade disposable polystyrene gel cassettes for an |X-Cell SureLock Mini-Cell gel electrophoresis apparatus, but could easily be adapted for other systems. Caution: wear gloves and protective clothing when preparing acrylamide gels! Acrylamide is a potent, cumulative neurotoxin.

• Tape bottom of gel cassettes, if necessary, and arrange upright in a rack
• Prepare a 12% resolving gel mixture in a small Erlenmeyer flask according to the recipe described in Electrophoresis Reagents.
• Add the TEMED and ammonium persulfate in that order, and mix thoroughly by swirling.
• Take up the mixture in a syringe, attach a narrow cannula or needle, and fill the gel cassettes with the gel mixture to within 2 cm of the top. Place 1-2 mL of remaining gel mixture in a microcentrifuge tube and cap tightly.
• Working quickly, gently layer 2-3 mm of distilled water on the top of the gel mixture in each cassette, using a syringe and needle.
• Allow the gel to polymerize for about 1 hr.¹ You may use the gel mixture in the microcentrifuge tube as a guide as to when polymerization is complete.
• Prepare a 4% stacking gel mixture in a small Erlenmeyer flask according to the recipe described in Electrophoresis Reagents.
• Add the TEMED and ammonium persulfate in that order, and mix thoroughly by swirling.
• Take up the mixture in a syringe, attach a narrow cannula or needle, and fill the gel cassettes with the gel mixture all the way to the top. Place 1-2 mL of remaining gel mixture in a microcentrifuge tube and cap tightly.
• Insert well-forming combs into each gel cassette, taking care not to trap bubbles under the comb.
• Allow the gel to polymerize for about 1 hr. You may use the gel mixture in the microcentrifuge tube as a guide as to when polymerization is complete.
• Gels may be used immediately, or can be stored in a zip-lock plastic bag with a damp paper towel. Gels can be stored for about 2-3 weeks before drying out.

Preparing samples for SDS-PAGE

• For clarified liquid samples, mix equal volumes of protein-containing solution and 2x SDS-PAGE loading buffer, prepared as described in Electrophoresis Reagents. Suggested final protein concentrations are about 0.1-1.0 ug/μL for homogeneous proteins or 1.0-10.0 ug/μL for protein mixtures. Heat sample to 95 °C for 5 min prior to loading.
• For whole cells (E. coli), pellet 1 mL of overnight culture in a microcentrifuge tube, and wash with 1.0 mL of 0.9% NaCl. To the washed pellet, add 25 μL of water and 25 μL of 2x SDS-PAGE loading buffer. Heat sample to 95 °C prior to loading, and centrifuge at 14000 xg for 3 min to pellet cell debris before loading.

Running and visualizing SDS-PAGE gels

The following protocol is typical for running an SDS-PAGE minigel. Wear gloves and protective clothing when initially handling and washing gels:

• Prepare 800 mL of 1x SDS-PAGE running buffer from 10x SDS-PAGE running buffer prepared as described in Electrophoresis Reagents.
• Remove the comb from a previously prepared gel cassette, and rinse the wells thoroughly with 1x running buffer to remove any unpolymerized acrylamide.
• Remove the tape from the bottom of the gel cassette, and place it into the gel appraratus.
  • The sample well opening should face the center compartment.
  • If running only one gel, place a dummy gel on the other side of the center well compartment.
• Fill the middle chamber with 1x running buffer above the top of the wells in the gel. Check for leaks into the outer compartment.
• Load up to 20 μL of sample into wells, as desired, using either a microsyringe or a micropipettor equipped with a gel-loading tip.
  • When loading samples insert gel-loading tip or microsyringe just below the top of the well. The sample will flow to the bottom of the well by gravity when dispensed. Do not poke the bottom of the well with the gel-loading tip or microsyringe
  • When analyzing protein fractions from FPLC, 5 μL samples are typical
• Fill the outer compartment with the remainder of the 1x running buffer, and affix the top of the gel apparatus.
• Attach the leads to a power supply and run gel(s) at a constant 250 V.² The gel run should be complete (as indicated by tracking dye migration to the bottom of the gel) in about 40-45 min.
• When the gel run is completed, switch off the power supply and disconnect the power leads.
• Remove the gel cassette from the apparatus, and using a knife, separate the halves of the gel cassette. Cut off the “foot” of the gel (which protrudes into the slot at the bottom) and the stacking gel.
  • Do not "slice" gels with the gel knife. "Punch" vertically with the gel knife to remove gel sections, making several punches as required to completely separate pieces. "Slicing" will tear the gels and may render them unusuable.
• Place the remaining portion of the gel in a container and complete cover with about 200-250 mL of staining solution prepared as described in Electrophoresis Reagents.
• Cover loosely, and microwave at full power for 1 min. Place on a shaker and slowly agitate for 10-15 min. Alternatively, the gel can be safely stained overnight at room temperature, if desired.
• When staining is complete, pour off the stain (it can be recycled and used many times) and rise the gel and its container with distilled water to remove excess stain solution.
• Add 200-250 mL of destaining solution prepared as described in Electrophoresis Reagents, cover loosely, and microwave at full power for 1 min. Place a few crumpled laboratory tissues into the container with the gel.³ Place on a shaker and slowly agitate until destaining is adequate to visualize bands. You may have to repeat the destaining cycle to completely destain the gel. Typically only 20-30 min is required per destaining cycle.

Drying SDS-PAGE gels

Gels are conveniently air-dried between membranes (Dry-Ease, Invitrogen) for permanent storage or imaging:

• Soak two drying membranes in distilled water.
• Place one half of rack and one soaked drying membrane on drying pedestal. Ensure the feet of the half-rack are pointing down. Soak liberally with water.
• Pour off destain solution from gel, and place gel on the drying membrane. Soak liberally in distilled water and remove bubbles from underneath gel with gentle pressure.
• Apply second soaked drying membrane on top of gel, taking care to remove all bubbles with gentle pressure.
• Assemble top half of drying rack to hold gel sandwich, matching rack feet. Use clips on all four sides to secure the assembly.
• Stand assembly upright and dry overnight at room temperature.
• Dried gels can conveniently imaged using a color flatbed scanner, and images analyzed using ImageJ.

Non-denaturing gel electrophoresis

Non-denaturing gel electrophoresis can be carried out in a manner directly analogous to SDS-PAGE, by simply omitting the denaturants and reducing agents from the gels, running buffer, and loading buffer. The required changes in the SDS-PAGE protocol are noted below:

• When preparing separating or stacking gel solutions, omit SDS from the recipe given in Electrophoresis Reagents.
• Running buffer should be prepared without SDS, but otherwise as given by Electrophoresis Reagents.
• 2x Loading buffer should be prepared without SDS or β-mercaptoethanol, but otherwise as in Electrophoresis Reagents
• Samples should not be heated prior to loading on the gel.
• Exact run times may vary from 30 min to 2 hr depending on the sizes and charges of proteins separated.

Analyzing gels using ImageJ

ImageJ can be used to quantify expression levels of recombinant proteins in crude lysates. There is a built-in tool in ImageJ to perform densitometry measurements of electrophoresis lanes. Instructions follow:

• Open an electrophoresis gel image in ImageJ. (A flatbed scan of a gel against a white background is ideal.)
• Using the rectangular selection tool, drag a thin rectangle along the lane to be quantified
• Choose Analyze...Gels...Select first lane...
• Choose Analyze...Gels...Gel analyzer options and select Invert Peaks, Label with percentages, and Uncalibrated OD.
• Choose Analyze...Gels...Plot lanes. A plot window will appear.
• Select the Line tool and draw a basline along the bottom of the densitometry trace. Additionally, draw vertical lines to divide the densitometry trace into segments of interest. Typically, the entire trace is divided into three segments: the peak of interest, everthing that comes before it, and everything that comes after it. Ensure that each segment is fully enclosed.
• Select the Magic Wand tool and click once within each of the densitometry trace segments. A separate window will appear with area measurements within each segment.
• Choose Analyze...Gels...Label Peaks and area percentages will be added to the plot

Notes

1. If the gel is insufficiently gelled after 1 hr, it will be necessary to begin again. To increase the rate of polymerization, add 10-20% more TEMED or ammonium persulfate to the gel mixture. If the mixture instead gels too quickly, decrease the TEMED or ammonium persulfate by 10-20%.
2. The literature suggests using a lower voltage, but this results in unnecessarily long run times. No degradation in performance is noted for standard tris-glycine SDS-PAGE gels at 250 V.
3. The tissues help absorb the staining dye, and speed the destaining procedure.

DNA sequencing sample preparation

Plasmids DNA can be conveniently sequenced using a dye-terminator-based cycle sequencing method. We are currently using the ABI Prism® Big Dye™ Terminator Cycle Sequencing Ready Reaction Kit supplied by PE Biosystems, in conjunction with an ABI 310 capillary electrophoresis sequencing system. Sequencing reactions can be carried out in any heated-top thermal cycler with adjustable temperature ramp times.

BigDye 3.1 Cycle Sequencing Guide

Cycle sequencing protocol

The basic protocol for generating sequencing fragments has been slightly modified to conserve reagents:

• For each sequence, pipet into a 0.5 mL PCR tube:
  • 4 μL terminator reaction ready mix¹
  • 2 μL 5x dilution buffer
  • 2-4 μL oligonucleotide primer (1.6 pmol/μL).² Please note concentration and read footnote.
  • 4-10 μL miniprep plasmid template (approx. 500 ng)
  • water to 20 μL
• Mix and centrifuge
• Perform 25 cycles as follows:³
  • 96 °C for 30 s
  • 50 °C for 15 s
  • 60 °C for 4 min
• At end of cycling hold at 4 °C
• Spin down sample prior to purifying extension products

Purifying extension products

The dye terminators are removed from the cycle-sequencing extension products by one of two methods.

Gel exclusion using Sephadex G-50

• Hydrate a Centri-Sep (Princeton Separations P/N CS-901) spin column by adding 0.8 mL water; allow to hydrate for 2 hr at room temperature.⁴
• Tap the column to settle gel and remove bubbles. Add a little more water if necessary.
• Remove upper, then lower end caps; allow column to drain completely by gravity.
• Insert column into wash tube, spin at 600 xg for 2 min to remove interstitial fluid.
• Insert column into a 1.5 μL microcentrifuge tube.
• Carefully pipet extension reaction mixture on to center of gel material.
• Spin at 600 xg for 2 min.
• Dry the collected sample in a vacuum centrifuge for 45 min at 60ºC, or until dry.⁵
• Resuspend dried sample in 10-20 μL of TSR buffer; vortex and spin.
• Denature at 95 °C for 2 min, vortex, spin, and chill on ice until loaded into the ABI 3100.

Zymo Research ZR Cleanup kit

This method is highly recommended, and is very quick. We routinely get 700-1200 bp reads from our cycle sequencing protocol using this cleanup method. Spin columns can be recycled if desired.⁶

• Add 240 μL of Sequencing Binding Buffer to up to 20 μL of sequencing reaction. Mix gently.
• Transfer entire mixture to a Zymo-Spin IB cleanup column placed in a collection tube
• Centrifuge at 15,000 xg for 30 seconds
• Add 300 μL Sequencing Wash Buffer to spin column
• Centrifuge at 15,000 xg for 30 seconds and discard flow-through. (Collection tube may be re-used later if desired)
• Place spin column in a 1.5 mL microcentrifuge tube
• Add 15 μL of water directly onto the membrane of the spin column. Let stand for a minute.
• Centrifuge at 15,000 xg for 30 seconds to collect DNA.
• This sample may be loaded directly into the ABI 3100 sequencer.

Notes

1. Store at –20 °C. Do not heat to melt; thaw on ice. This solution includes dye terminators, dNTPs, AmpliTaq DNA polymerase, pyrophosphatase, MgCl₂, and Tris buffer, pH 9.0. Store at –20 °C.
2. The 1.6 pmol/μL oligonucleotide solution should be prepared fresh by diluting the working stock 50 pmol/μL solution 1:31. The 1.6 pmol/μL solution should be discarded immediately after use. The use of 4 μL of diluted oligonucleotide primer (6.4 pmol) may result in up to 2x stronger signal in the sequencing reaction, especially if there are more than 500 ng of plasmid template available. Otherwise 2 μL of oligonucleotide primer (3.2 pmol) is recommended.
3. Ramp time should be set at 1°C/s for optimal results
4. Hydrated columns can be stored in the refrigerator for 1-2 days; warm to room temperature before use. Columns can also be re-used. Immediately after use, discard Sephadex from column. Rinse thoroughly with water and centrifuge 700 μL of water through the column. After columns have air-dried, fill with 0.8 mL hydrated Sephadex G-50 (10 g per 150 mL water), let drain, and centrifuge as you would for new Centri-Sep columns.
5. Do not over-dry! Dried samples may be stored in the freezer until ready for electrophoresis.
6. To recycle Zymo Research IB sping columns, add 500 μL 0.1% HCl and let stand for 30 seconds. Centrifuge at 15,000 xg for 30 seconds. Dry overnight at 37 °C before use. Columns may be regenerated 10 times or more if membrane integrity is maintained.

Verifying DNA Sequence

• Start the FinchTV ABI file reader program
• Drag your ABI file into the FinchTV box
• Find your promoter (NcoI is CCATGG) and delete everything preceding the start codon ATG (e.g. for NcoI eliminate through CC)
• Export sequence by clicking on Choose File...Export, label and save in desired folder.
• Using web browser, go to www.expasy.org and click "proteomics" and then click "translate" on the bottom right corner of the page to open the translate tool.
• Highlight the exported sequence and copy/paste it into the translate tool input box
• Choose the "include nucleotide sequence" option from the scroll down menu under the input box and then click on "translate".
• Compare each amino acid in the DNA sequence to that of the known gene sequence.