Hoefer HE 33 mini submarine electrophoresis unit User Manual

Page finder Mini submarine electrophoresis unit function . .

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Important information...

2

Unpacking...

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Specifications...

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Operating instructions...

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Care and maintenance...

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Troubleshooting...

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Notes, buffers, and volumes...

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Bibliography...

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Ordering information...

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Mini submarine electrophoresis unit function

Fig 1. Main components. (See Figure 2 for an illustration of the casting kit.)

color-coded leads connect electrodes in the unit base to the power supply.

electrode connectors (2)

The Hoefer™ HE 33 horizontal agarose unit is intended for rapid electrophoresis of small quantities of nucleic acids in agarose gels. A gel is cast in the gel caster, which holds one or two combs. (Eight different combs are available; a maximum of 32 samples can be run if two 16-well combs are used.) After the gel sets, the running tray is transferred to the platform of the horizontal unit. The base of the unit holds coolant that can be chilled before the run. This passive cooling capacity allows fast, high voltage runs.

lid assembly

running platform (supports the running tray)

fill the base with 50/50 ethylene glycol/water

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Important information • The safety lid must be in place before connecting the power leads to a power supply. • Turn all power supply controls off and disconnect the power leads before removing the safety lid. • Before the first use, fill the base with about 600 ml 50/50 ethylene glycol/water to prevent irreparable damage to the unit. (See instructions on page 4.) The base must be filled even if no cooling is required. • Do not use pure water, commercial anti-freeze, or any organic solvent to fill the base. Water expands as it freezes. Because the water is trapped in the base, it may crack. Organic solvents will cause irreparable chemical damage to the unit! • Do not chill the base below -20 °C (-4 °F). Chill the base in a bucket of ice, refrigerator, or in a freezer. • Do not operate with gel or buffer temperature above 50 °C. Prevent overheating by chilling the base prior to use. To prevent overheating during prolonged high-voltage runs, exchange the first base with an extra prechilled second base if one is available. Overheating will cause irreparable damage to the unit! • If this equipment is used in a manner not specified by the manufacturer, the protection provided by the equipment may be impaired. • Only accessories and parts approved or supplied by Hoefer, Inc. may be used for operating, maintaining, and servicing this product.

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This declaration of conformity is only valid for the instrument when it is: • used in laboratory locations, • used as delivered from Hoefer, Inc. except for alterations described in the User Manual, and • connected to other CE labeled instruments or products recommended or approved by Hoefer, Inc.

Unpacking Unwrap all packages carefully and compare contents with the packing list, making sure all items arrived. If any part is missing, contact your local sales office. Inspect all components for damage that may have occurred while the unit was in transit. If any part appears damaged, contact the carrier immediately. Be sure to keep all packing material for damage claims or for repacking should it become necessary to return the unit.

Specifications Max. voltage

500 V for 5 minutes or less

Max. wattage

15 W

Max. current

500 mA

Max. operating temp.

50 °C

Max. buffer volume

250 ml

Coolant required

≈600 ml 50/50 water/ethylene glycol

Gel size

7 × 10 cm

Environmental operating conditions

Installation category

II

Pollution degree

2

Dimensions

width × depth × height 24 × 13 × 7 cm (9.5 × 5.2 × 2.8 in.)

Weight (base, lid, leads only) Product certifications

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Indoor use: 4–40 °C Humidity up to 80% Altitude up to 2000 m

0.4 kg (0.9 lbs) EN61010–1, UL61010A-1, CSA C22.2 1010.1, CE Certified

Operating instructions Agarose gels are first prepared using the gel casting kit. Samples are then loaded into wells and electrophoretically separated. The fluorescent dye ethidium bromide can be added to the gel or electrophoresis buffer or both to track separation progress. After electrophoresis, the gel may be stained and photographed, blotted, or dried for autoradiography.

Fill the base with coolant Important! Do not fill the base with commercial antifreeze, organic solvents, or pure water.

Even if no cooling is required, it is important to fill the base with the proper coolant solution before the first use because the solution provides a necessary heat sink.

Note: It is not necessary to replace the coolant.

 Prepare 600 ml of 50/50 ethylene glycol/water. Optional: To help see wells more clearly while loading the sample, add a drop or two of soluble dye or food color to the coolant solution. Locate the two inlet holes in the top edge of the base. Fill the base cavity as full as possible with coolant using a 50-ml syringe or a pump.

 Push a gray rubber plug into each hole, taking care that the plug is securely seated.

 Place the prepared base in an ice bucket or into a refrigerator or freezer set no lower than -20 °C for about an hour before use. (The base will always be ready if you store it in the refrigerator or freezer.)

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Prepare solutions Caution: Ethidium bromide is a known mutagen. Always wear gloves when handling.

 Prepare 250 ml of running buffer. (Refer to p. 12 for recipes of commonly used electrophoretic running buffers.)

 Prepare the sample loading buffer. (Refer to p. 13 for a recipe and a table of volume requirements for each comb size.)



Fig 2. Gel casting kit. Approach the foam pad with one end of the running tray and then gently press the tray edge against the pad, compressing it enough to allow the opposite end of the running tray to drop fully into the casting tray before sealing against the foam pad.

Prepare approximately 7 ml agarose solution per mm of gel thickness. (For example, a 3-mm gel requires 0.3 cm × 7 cm × 10 cm = 21 ml) Dissolve agarose in running buffer, heat according to instructions accompanying the agarose, and allow the solution to cool to 50 °C before pouring into the casting tray. Optional: Add 0.5 µg/ml ethidium bromide to the gel solution to observe separation during electrophoresis.

UV-transparent running tray (cast the gel on this tray, then transfer gel to the horizontal unit base for electrophoresis.)

gel casting tray foam pads (2)

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Cast the gel  Install the running tray. Firmly grasp the casting tray with one hand. With the other hand, place one end of the running tray against the foam pad at the bottom edge, press the tray against the pad, and then lower it to rest on the bottom of the casting tray, seating the other end of the tray against the opposite foam pad.

 Prepare the comb(s). Fit the two slots in the comb between the (loosened) thumb screw heads and the comb back. Tighten the screws until the comb is just supported. Seat the comb assembly on the rim of the casting tray and adjust the bottom of the comb so that it is about 1.0 mm from the running tray. Tighten the screws to secure the comb. To run twice as many samples, prepare two combs.

 Remove the comb assembly. Place the casting assembly on a leveling surface and level, using the spirit level on the running tray as a guide.

 Pour the agarose solution (cooled to 50 °C) into the casting tray. Orient the comb assembly so that the comb faces the nearest foam pad and seat it on the tray rim. Check that the comb is vertical to prevent well shape distortions. To run twice as many samples, place the second comb assembly in the center of the tray. Allow a minimum of 30 minutes for the gel to set.

 Once the gel is set, remove the comb carefully. Partially lift and slightly tilt the comb at one end and then slowly withdraw it from the gel. (Pulling the comb straight up creates a vacuum in the wells that may lift the gel out of the tray.)

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

Fig. 3. A comb back, which fits onto the rim of the casting tray, positions the comb in the gel. Two screws hold the adjustable comb.

Remove the running tray and gel by grasping the handles of the tray and pressing against one of the foam pads. Once the tray clears the opposite pad, lift it out. Transfer the running tray and gel to the chilled base.

For twice as many wells, a second comb can be placed in the center of the gel.

comb comb back

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screws (2)

The electrophoresis run Refer to the notes, buffers, and volumes section for additional information and guidelines.

 Chill the base before use, especially when higher voltage settings will be used or when the separation will require more than 30 minutes. Note: To monitor separation progress, either add 0.5 µg/ml (final conc.) of ethidium bromide to the running buffer now, or add 50 µg/ml (final conc.) ethidium bromide to the sample buffer. To visualize progress, turn off the power supply, remove the lid assembly, and hold a portable UV lamp near the gel. Caution: Ethidium bromide is a known mutagen. Always wear gloves when handling. Wear UV safety goggles and protect skin when using a UV lamp.

Adding ethidium bromide to the running or sample buffer slows migration slightly. Detection by this method is not as sensitive as staining and viewing on a transilluminator. (See DNA detection, p. 15.)

 Fill both buffer chambers with running buffer until the buffer is ~1 mm deep over the gel. (This requires about 220 ml.)

 Note: If no dye was added to the coolant, place the base on a dark background to see the wells more easily.

Note: At the maximum setting the unit begins overheating as soon as the chilled base reaches ambient temperature. If overheating is not controlled, the gel will melt and/or the base of the unit will warp!

Load the samples. Add sample to 5X sample loading buffer and mix (1/5 of the final volume is loading buffer, see p. 14). Use a micro-pipette to load each sample, taking care to avoid puncturing the well bottom or entrapping any bubbles.

 Place the lid so that the cathode (–, black lead) is at the end nearest the sample well. (Nucleic acid samples migrate toward the anode, +, red lead.) Connect the color-coded leads (red to red, and black to black) to an approved power supply, such as the EPS 2A200. Set the voltage level and timer (if available) according to the degree of resolution sought.

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Quick, high-voltage runs Certain applications, such as screening samples or checking sample purity, can be accomplished quickly under high voltage conditions. Chill the base (-20 °C) and limit the run to 5 minutes or less at 500 V.

Slower, lower voltage runs Note: To calculate the voltage gradient, divide the voltage setting by the distance between the electrodes (12.7 cm).

A voltage gradient of 12 V/cm (150 V) separates 0.1 to 23 kb fragments of a Hind III digest of λ DNA in 30 to 40 minutes (using 1% agarose gel and 0.5X TBE running buffer). Alternatively, using the same solutions, this sample could be run at 24 V/cm (300 V) with acceptable band resolution in 20 to 30 minutes. Chill the base before use.

Table 1: Voltage settings and recommended run settings† voltage (V)

gradient (V/cm)

time (min)

500

40

5*

400

31

10*

300

24

20*

200

16

30 to 40

150 12 30 to 60 *For rapid runs of 20 minutes or less, use 0.5X TBE and chill the base to -20 °C before use. †

Voltage and times are for 1% Agarose NA, 0.5X TBE and a chilled base.

After the separation  Turn off the power supply, disconnect the leads, and remove the lid.

 If no ethidium bromide was added to the gel or sample before the run, stain the gel in a solution of 0.5 to 1.0 µg/ml ethidium bromide in water or buffer.

 Clean the unit as described below.

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Care and maintenance Cleaning Important! Never autoclave any component of the electrophoresis unit or casting kit.

After each use, clean the unit with a mild detergent and water, rinse thoroughly with distilled water, and allow to air dry. Never use abrasive cleansers. Do not expose the unit to solutions or vapors of aromatic or halogenated hydrocarbons, ketones, esters, alcohols (over 30%), or concentrated acids (over 25%). To reduce DNase and RNase contamination, soak the buffer chamber or casting kit for 10 minutes in a 3% hydrogen peroxide (H2O2) solution, then rinse thoroughly with DEPCtreated, autoclaved, deionized water. (Sambrook, et al. 1:7:40)

Replacing foam pads Remove worn foam pads. Peel off the adhesive cover on a new foam pad. Align the pad so that it will rest on the bottom of the tray along a short (7-cm) side, adhesive side toward the inside wall, and then press it in place. Repeat with second pad on the wall opposite the first pad.

Replacing the electrode It is recommended that electrodes be replaced only by Hoefer technicians. Call your local representative for advice.

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Troubleshooting problem

solution

Deformed sample well

Allow the gel to set for a minimum of 30 minutes and make sure it is at room temperature before removing the comb. When removing the comb, hold it at a slight angle and lift very slowly to prevent the gel from breaking. Take care to not damage the well with the pipet while loading the sample; aim for the center of the well and do not puncture the bottom with the pipet tip.

Samples not running along a straight path

If a comb or running tray is warped, replace. Reduce the voltage. Choose a buffer with the appropriate ionic strength and buffering capacity. (The buffering capacity of TBE, for example, is higher than that of TAE.) If the buffer is depleted, stop the run, remove the lid, and pipette the buffer from each chamber into the opposite chamber to replenish the buffer. If the gel is uneven, level the casting tray before pouring the gel.

Double-banded pattern

The comb must be vertical to prevent well shape distortion. Decrease the buffer level to 1 mm above the top of the gel, to reduce the vertical temperature gradient.

Poor band resolution

Add Ficoll™, glycerol, or sucrose to the sample loading buffer to ensure that the sample sinks to the bottom of the well. (Ficoll is preferred.) Make sure the sample is completely dissolved. Reduce the voltage. Reduce the sample concentration. Reduce the sample volume. At least 1 mm of gel below the bottom of the comb is required to prevent samples from leaking out of the well bottom. Reduce the salt concentration of the sample. Check enzyme activity; the sample may require longer digestion or a different restriction buffer. Prepare fresh sample if you suspect nuclease contamination. Choose agarose with a low endosmosis value.

Foam pads peel off

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Install the running tray as as described on page 5; do not press straight down into place.

Notes, buffers, and volumes Agarose gel electrophoresis notes Agarose gel electrophoresis can be used to separate DNA fragments as small as 0.1 kb or less. Polyacrylamide gels are usually used for fragments smaller than 1 kb.

DNA mobility Suggested agarose concentration for separating fragments of various sizes is given in Table 2 below. Other factors affecting separation results include the running buffer, voltage setting, temperature, conformation, and the presence of ethidium bromide. Special agaroses are available that can extend resolution ranges. A common standard is a Hind III digest of lambda phage, which gives eight fragments ranging in size from 0.1 to 23 kb pairs. For good resolution, run 45 minutes on a 10-cm long, 1% agarose gel in 0.5X TBE gel at 150 V. Table 2: Agarose concentrations for separating DNA fragments of various sizes agarose (%)

effective range of resolution of linear DNA fragments* (kb)

0.5

1.0–30

0.7

0.8–12

1.0

0.5–10

1.2

0.4–7

1.5

0.2–3

*Current Protocols in Molecular Biology, p 2.5.2 (1993)

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RNA mobility RNA can also be separated on the basis of size. To avoid anomalies due to secondary structure, RNA is denatured either before or during electrophoresis. For example, RNA fragments previously denatured with glyoxal and dimethylsulfoxide can be separated on neutral agarose gels, or RNA can be fractionated on agarose gels containing methylmercuric hydroxide or formaldehyde. RNA samples usually require longer runs or buffers that are easily depleted, and so require circulation. The Hoefer HE 100 horizontal unit is recommended for this application rather than the HE 33.

Running buffers for DNA in agarose gels Important! Do not adjust the pH of these buffers once they are prepared according to the recipe!

Recipes for the two most commonly used running buffers for DNA electrophoresis are listed below. The ionic strength of these buffers is appropriate for the application; do not adjust the pH of these buffers once they are prepared according to the recipe! 1. 10X Tris-borate-EDTA (TBE) stock buffer† (0.89 M Tris, 0.89 M boric acid, 20 mM EDTA, pH ∼8.2, 1000 ml) Tris base (FW 121.1)

0.89 M

108.0 g

Boric acid (FW 61.8)

0.89 M

55.0 g

EDTA solution (0.5 M, pH 8.0, soln. 3)

0.02 M

Deionized H2O

40.0 ml to 1000.0 ml

Stir. Do not adjust pH. Before use dilute either to: 0.5X, to yield 45 mM Tris base, 45 mM boric acid, and 1 mM EDTA. This dilution is often used because current remains low, resulting in less heat.

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-or- 1X, to yield 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA.

2. 10X Tris-acetate-EDTA (TAE) stock buffer† (0.4 M Tris, 0.2 M acetic acid, 10 mM EDTA, pH ~8.4, 1000 ml) Tris base (FW 121.1)

0.40 M

48.4 g

Acetic acid (99.5%)

0.20 M

11.4 ml

EDTA solution (0.5 M, pH 8.0, soln. 3)

0.01 M

Deionized H2O

20.0 ml to 1000.0 ml

Stir. Do not adjust pH. Dilute to 1X before use to yield 40 mM Tris base, 20 mM acetic acid, and 1 mM EDTA.

3. EDTA solution (ethylenediamine tetraacetic acid)† (0.5 M, pH 8.0, 100 ml) Na2EDTA·2H2O, (FW 372.2) †

Modified from Sambrook, J., Molecular Cloning: A Laboratory Manual, p. B.23 (1989). See also Current Protocols in Molecular Biology, p. A.2.1 (1993).

Deionized H2O NaOH (10 M) to pH 8.0 Deionized H2O

0.5 M

18.6 g to 70.0 ml ~5.0 ml to 100.0 ml

Sample loading buffer Loading buffer (5X, 25% Ficoll 400, 0.25% Bromophenol blue†,10 ml) Deionized H2O

to 7.0 ml

Ficoll 400

2.5 g

Bromophenol blue (FW 691.9)

25.0 mg

Deionized H2O

to 10.0 ml

Add to sample in proportion so that 1/5 of the final volume is loading buffer. (Loading buffer increases solution density.) Note 1: Sucrose or glycerol may be used instead of Ficoll 400.

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Note 2: Xylene cyanol (0.25%), which migrates more slowly than bromophenol blue, can be added as an additional marker if desired. The agarose concentration determines the position of the dye bands relative to a polynucleotide. †

Tracking dyes may be omitted to eliminate obscuring and dragging effects caused by comigration with smaller nucleic acids.

Well volume Table 3: Comb specifications comb code no.

no. of wells

well thickness width (mm) (mm)

sample vol. per 1 mm depth (µl)

HE31A-P-1.0

1 prep/2 ref

1.0

44/6

44/6*

HE31A-P-1.5

1 prep/2 ref

1.5

44/6

66/9*

HE31A-8-1.0

8

1.0

6.5

6.5

HE31A-8-1.5

8

1.5

6.5

9.7

HE31A-12-1.0 12

1.0

3.9

3.9

HE31A-12-1.5 12

1.5

3.9

5.8

HE31A-16-1.0 16

1.0

2.6

2.6

HE31A-16-1.5 16 1.5 2.6 3.9 *The preparative combs form two reference wells (for MW standards), one on each side of the preparative well. The first number is sample volume/mm in the preparative well; the second is volume/mm in the reference well.

DNA detection Caution! Ethidium bromide is a known mutagen. Always wear gloves when handling. Wear UV safety goggles and protect skin when using any UV light source. Note: Ethidium bromide slows DNA migration by about 15%. Note: Minimize the staining time to prevent small nucleic acid fragments from diffusing out of the gel.

DNA can be detected either by the fluorescence of bound ethidium bromide or by autoradiography of radiolabeled DNA. Ethidium bromide (0.5 µg/ml) can be added to running buffer to monitor sample progress because the dye’s fluorescence under a UV lamp reveals band location. (To check progress, turn off the power supply and remove the lid of the agarose unit. Hold a portable UV-lamp near the running tray. Replace the lid and turn on the power again to resume electrophoresis.) Alternatively, after electrophoresis, stain the gel in an ethidium bromide solution (0.5 µg/ml

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H2O) for 15 to 60 minutes and then view or photograph the sample on a UV transilluminator. To photograph the gel, either place the running tray on the transilluminator surface or slide the gel onto the surface for maximum exposure. (The running tray is 95% transparent to 302-nm light and 40% transparent to 254 nm light.) View the sample under 366-nm UV light or reduced intensity 302-nm UV light to reduce photonicking. To reduce the background fluorescence of unbound ethidium bromide, the gel can be destained by soaking it for 5 minutes in 0.01 m MgCl2, or for 1 hour in 0.001 M MgSO4. Destaining makes it easier to detect small quantities (less than 10 ng) of DNA. (Sambrook, section 6.15)

Bibliography Ausubel, et al., (eds). Current Protocols in Molecular Biology. Greene Publishing and Wiley-Interscience. New York (1993). Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press (1989).

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Ordering information All quantities are 1 except where indicated.

Basic unit and kit

code no.

Mini Submarine Electrophoresis Unit, basic. Includes gel running tray, gel casting tray and bubble level. (Order comb and comb back separately.)

HE33B

Mini Submarine Electrophoresis Unit, kit. Same as above plus one 8-well, 1.5 mm thick comb, comb back, and screws.

HE33-8-1.5

Replacement parts Buffer chamber assembly

HE30

Lid with high voltage leads

HE36

Fill plug, bottom

HE38

Fill plug, top

HE38TP

Gel running tray, UVT, 7 × 10 cm

HE42-10

Casting tray, 7 × 10 cm

HE45-10

Gel casting kit with gel casting tray and running tray, 7 × 10 cm

HE47-10

Foam gaskets (pk/2)

HE48

High voltage leads

SE6056-HV

Electrode replacement kit

HE39

Bubble level

SER11

SE6056-HV

HE42-10 HE36

HE30

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HE48

HE45-10

Combs Specify thickness and number of wells, as tabulated below. (Order a comb back separately.) thickness (mm)

no. of wells

code no.

1.0

Preparative

HE31A-P-1.0

1.0

8

HE31A-8-1.0

1.0

12

HE31A-12-1.0

1.0

16

HE31A-16-1.0

1.5

Preparative

HE31A-P-1.5

1.5

8

HE31A-8-1.5

1.5

12

HE31A-12-1.5

1.5

16

HE31A-16-1.5

Comb back with 2 screws

HE31-BK

Replacement screws for comb backs (pk/12)

HE31-S

Companion products Hoefer EPS 2A200 power supply

PS2A200

MacroVue UV-20 Transilluminator 115 V~

UV20-115V

230 V~

UV20-230V

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