Hoefer HE33 User manual

user
manual
nucleic acid electrophoresis
um
HE33-IM/Rev. H0/05-04
Hoefer HE 33
mini submarine electrophoresis unit

Page  nder
Mini submarine electrophoresis unit function
. .
1
Important information
. . . . . . . . . . . . . . . . . . .
2
Unpacking
. . . . . . . . . . . . . . . . . . . . . . . . . . .
4
Speci cations
. . . . . . . . . . . . . . . . . . . . . . . .
4
Operating instructions
. . . . . . . . . . . . . . . . . .
5
Care and maintenance
. . . . . . . . . . . . . . . . .
11
Troubleshooting
. . . . . . . . . . . . . . . . . . . . . .
12
Notes, buffers, and volumes
. . . . . . . . . . . . .
13
Bibliography
. . . . . . . . . . . . . . . . . . . . . . . . .
17
Ordering information
. . . . . . . . . . . . . . . . . . .
18

•
pii

•
p1
Fig 1.
Main components.
(See Figure 2 for an illustration
of the casting kit.)
electrode connectors (2)
running platform
(supports the running tray)
color-coded leads connect
electrodes in the unit base to
the power supply.
lid assembly
 ll the base with 50/50
ethylene glycol/water
Mini submarine electrophoresis
unit function
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 avail-
able; 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 plat-
form 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.

•
p2
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  rst use,  ll 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  lled even if no cooling is
required.
• Do not use pure water, commercial anti-freeze, or
any organic solvent to  ll 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  rst 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 speci ed
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.

•
p3
Informations importantes
• Le couvercle de sécurité doit être en place avant
de brancher les prises au générateur.
• Eteindre le générateur et débrancher les prises
avant d’enlever le couvercle de sécurité.
• Avant la premiére utilisation, remplir le socle
avec environ 600 ml 50/50 d’éthylène glycol et
d’eau a n de ne pas endommager l’instrument.
(Voir page 4 du mode d’emploi). Le socle doit
être rempli même si le refroidissement n’est pas
nécessaire.
• Ne pas utiliser seulement de l’eau, de l’anti-
gel commercial ou tout autre solvant organique
pour remplir le socle. L’eau augmente en volume
lorsqu’elle gêle. Du fait que l’eau est enfermée,
le socle peut se fendre. Les solvants organiques
peuvent causer des dommages chimiques
irréparables.
• Ne pas refroidir le socle en dessous de -20 °C
(-4 °F). Refroidir le socle dans un seau rempli de
glace ou un dans un réfrigérateur.
• Ne pas utiliser avec un gel ou un tampon à
plus de 50 °C. Eviter le surchauffement en
refroidissant le socle avant l’emploi. A n d’éviter
le surchauffement durant de longues utilisations
sous haut voltage, échanger si possible, le
socle avec un second socle déjà refroidi.Un
surchauffement peut causer des dommages
irréparables à l’instrument.
• Si l’instrument n’est pas utilisé en conformité
avec les recommandations du fabriquant, les
protections de sécurité qui équipent cet appareil
peuvent être rendues inéf caces.
• Seulement les accessoires et piéces detachées
approuvés ou fournis par Hoefer, Inc. sont
recommandés pour l’utilisation, l’entretien et
réparation de cet appareil.

•
p4
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 of ce. 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.
Speci cations
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
Indoor use: 4–40 °C
Humidity up to 80%
Altitude up to 2000 m
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)
0.4 kg (0.9 lbs)
Product certi cations
EN61010–1, UL61010A-1,
CSA C22.2 1010.1, CE
Certi ed
This declaration of confor-
mity 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.

•
p5
Operating instructions
Agarose gels are  rst prepared using the gel cast-
ing kit. Samples are then loaded into wells and
electrophoretically separated. The  uorescent
dye ethidium bromide can be added to the gel or
electrophoresis buffer or both to track separa-
tion progress. After electrophoresis, the gel may
be stained and photographed, blotted, or dried
for autoradiography.
Fill the base with coolant
Even if no cooling is required, it is important to  ll
the base with the proper coolant solution before the
 rst use because the solution provides a necessary
heat sink.

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.)
Important!
Do not  ll the base
with commercial antifreeze,
organic solvents, or pure
water.
Note:
It is not necessary to
replace the coolant.

•
p6
Prepare solutions

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.)

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.
Caution:
Ethidium bromide is a
known mutagen. Always wear
gloves when handling.
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.
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)

•
p7
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.)

•
p8

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.
comb back
comb
screws (2)
Fig. 3.
A comb back, which  ts
onto the rim of the casting tray,
positions the comb in the gel. Two
screws hold the adjustable comb.
For twice as many wells, a second
For twice as many wells, a second
comb can be placed in the center
comb can be placed in the center
of the gel.
of the gel.

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 ( nal conc.) of ethidium bromide to the running
buffer now, or add 50 µg/ml ( nal 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.
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.)

Load the samples.
Add sample to 5X sample loading buffer and mix (1/5
of the  nal 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.
•
p9
Caution:
Ethidium bromide
is a known mutagen. Always
wear gloves when handling.
Wear UV safety goggles and
protect skin when using a UV
lamp.
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!

•
p10
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
A voltage gradient of 12 V/cm (150 V) separates 0.1
to 23 kb fragments of a
Hin
d 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
gradient
time
(V)
(V/cm)
(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.
Note:
To calculate the voltage
gradient, divide the voltage
setting by the distance
between the electrodes
(12.7 cm).

Care and maintenance
Cleaning
After each use, clean the unit with a mild deter-
gent 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 hydrocar-
bons, 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 (H
2
O
2
)
solution, then rinse thoroughly with DEPC-
treated, 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  rst
pad.
Replacing the electrode
It is recommended that electrodes be replaced
only by Hoefer technicians. Call your local
representative for advice.
•
p11
Important!
Never autoclave
any component of the
electrophoresis unit or casting
kit.

•
p12
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 punc-
ture 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 buff-
ering 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 cham-
ber 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
™
, 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
Install the running tray as as described on page 5; do not
press straight down into place.
Troubleshooting

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 sepa-
rating fragments of various sizes is given in
Table 2 below. Other factors affecting separa-
tion results include the running buffer, volt-
age setting, temperature, conformation, and
the presence of ethidium bromide. Special
agaroses are available that can extend resolu-
tion ranges.
A common standard is a
Hin
d 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
effective range of resolution
agarose (%)
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)
•
p13

•
p14
RNA mobility
RNA can also be separated on the basis of size.
To avoid anomalies due to secondary struc-
ture, RNA is denatured either before or during
electrophoresis. For example, RNA fragments
previously denatured with glyoxal and dimeth-
ylsulfoxide can be separated on neutral agarose
gels, or RNA can be fractionated on agarose gels
containing methylmercuric hydroxide or formal-
dehyde.
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
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
40.0 ml
Deionized H
2
O
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.
Important!
Do not adjust the
pH of these buffers once they
are prepared according to the
recipe!

•
—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
20.0 ml
Deionized H
2
O
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)
Na
2
EDTA·2H
2
O, (FW 372.2)
0.5 M
18.6 g
Deionized H
2
O
to 70.0 ml
NaOH (10 M) to pH 8.0
~5.0 ml
Deionized H
2
O
to 100.0 ml
Sample loading buffer
Loading buffer
(5X, 25% Ficoll 400, 0.25% Bromophenol blue
†
,10 ml)
Deionized H
2
O
to 7.0 ml
Ficoll 400
2.5 g
Bromophenol blue (FW 691.9)
25.0 mg
Deionized H
2
O
to 10.0 ml
Add to sample in proportion so that 1/5 of the  nal
volume is loading buffer. (Loading buffer increases
solution density.)
Note 1:
Sucrose or glycerol may be used instead of
Ficoll 400.
•
•
•
•
p15
• p15 •
p15 p15p15
†
Modi ed from Sambrook, J.,
Molecular
Cloning: A Laboratory Manual,
p. B.23
(1989). See also Current Protocols in
Molecular Biology, p. A.2.1 (1993).

•
p16
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 speci cations
well
sample vol.
comb
no. of
thickness
width
per 1 mm
code no.
wells
(mm)
(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
 rst number is sample volume/mm in the preparative well;
the second is volume/mm in the reference well.
DNA detection
DNA can be detected either by the  uorescence
of bound ethidium bromide or by autoradiogra-
phy of radiolabeled DNA.
Ethidium bromide (0.5 µg/ml) can be added
to running buffer to monitor sample progress
because the dye’s  uorescence 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
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.