Bio-Rad Sequi-Gen GT Sequencing Cell User Manual
Sequi-Gen
Sequi-Gen®GT
Nucleic Acid
Electrophor esis Cell
Instruction Manual
Catalog Numbers
165-3860, 165-3861,
165-3862 and 165-3863
For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)
Sequencing Cell
®
GT
Note
To insure best performance from the Sequi-Gen GT electrophoresis system, become fully acquainted with these operating instructions before using the cell. Bio-Rad recommends that you first read these
instructions carefully. Then assemble and disassemble the cell completely without casting a gel. After
these preliminary steps, you should be ready to cast and run a gel.
Bio-Rad also recommends that all Sequi-Gen GT components and accessories be inspected for damage, cleaned as recommended in this manual, and rinsed thoroughly with distilled water before use.
Record the following for you records:
Warranty
Bio-Rad Laboratories warrants the Sequi-Gen GT electrophoresis system against defects in
materials and workmanship for 1 year. If any defects occur in the instrument during this warranty period, Bio-Rad Laboratories will repair or replace the defective parts free. The following defects, however, are specifically excluded:
1. Defects caused by improper operation
2. Repair or modification done by anyone other than Bio-Rad Laboratories or an authorized
agent.
3. Use of fittings or other spare parts supplied by anyone other than Bio-Rad Laboratories.
4. Damage caused by accident or misuse.
5. Damage caused by disaster
6. Corrosion due to use of improper solvent or sample
This warrant does not apply to parts listed below:
1. Platinum wire
2. Glass plates
For any inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the model
and serial number of your instrument.
Model
Catalog No.
Date of Delivery
Warranty Period
Serial No.
Invoice No.
Purchase Order No.
Table of Contents
Page
W arranty Information ........................................................................Inside Front Cover
Section 1 General Information....................................................................................1
1.1 Introduction to Sequi-Gen GT DNA Electrophoresis Cell .......................................1
1.2 Specifications .............................................................................................................5
Section 2 Description of Major Parts.........................................................................6
2.1 Sequi-Gen GT Parts ...................................................................................................6
2.2 Gel Reagents and Electrophoresis Buffers ................................................................7
2.3 Electrical Path.............................................................................................................8
Section 3 Cleaning and Maintenance.........................................................................8
3.1 Cleaning and Siliconizing Plates................................................................................8
3.2 Cleaning Plastic Parts.................................................................................................9
Section 4 Operating Instruction................................................................................10
4.1 Before Assembly......................................................................................................10
4.2 Assembling the Glass Plate Sandwich.....................................................................10
4.3 Casting the Gel.........................................................................................................12
4.4 Preparing for Operations..........................................................................................17
4.5 Loading the Gel........................................................................................................18
4.6 Gel Electrophoresis..................................................................................................19
4.7 Disassembly..............................................................................................................20
Section 5 Troubleshooting Guide..............................................................................22
5.1 Operational Troubleshooter .....................................................................................22
5.2 DNA Sequencing Artifacts ......................................................................................23
Section 6 Equipment and Accessories......................................................................26
6.1 Sequi-Gen GT Nucleic Acid Electrophoresis Cells and Accessories.....................26
6.2 Electrophoresis Reagents.........................................................................................30
6.3 Power Supplies and Slab Gel Dryers.......................................................................31
6.4 DNA Template Purification, Sequencing, and Cloning Products...........................31
6.5 Liquid Handling .......................................................................................................32
Section 7 Appendix A- Applications.........................................................................32
7.1 DNA Sequencing Checklist.....................................................................................32
7.2 Standard Gel Protocol ..............................................................................................33
7.3 Gel Drying Autoradiography...................................................................................34
7.4 Applications for Sequi-Gen GT Nucleic Acid Electrophoresis Cell ......................34
7.5 Suggested Reading...................................................................................................35
Section 1
General Information
1.1 Introduction to the Sequi-Gen*GT Nucleic Acid Sequencing Cell
The Sequi-Gen GT cell is a modular electrophoresis cell capable of separating nucleic
acids with single base-pair resolution, using a vertical slab gel format. This manual tells you
how to operate and care for your new Sequi-Gen GT cell. Read Sections 1 through 3 before
attempting to assemble the cell. The remainder of the manual gives you detailed procedures,
a troubleshooting guide, and parts lists.
The Sequi-Gen GT cell employs a simple design that provides maximum resolution with
high reproducibility, while eliminating the temperature artifacts which often occur in sequencing gels. Some of the unique features of this sequencing cell are the gel casting method,
durable construction, modular components, and ease of operation, which make this the most
advanced DNA sequencing cell available.
Note:
This manual contains instructions for the Sequi-Gen GT electrophoresis systems only.
Prior to the release of the Sequi-Gen GT systems, Bio-Rad supplied two similar sequencing electrophoresis cell systems: the original Sequi-Gen cell and the Sequi-Gen II cell. This
manual does not provide information on these systems. Contact your Bio-Rad representatives for information on the original Sequi-Gen and the Sequi-Gen II systems.
* US Patent number 4,663,015 issued to Bio-Rad Laboratories.
Safety
The Sequi-Gen GT cell has safety features to protect the operator from injury. These
features include:
• Interlocking safety lids to prevent high voltage buffer shock
• Permanently sealed upper buffer chamber to prevent leaks and arcing
• Plastic components made from self-extinguishing material
• Full-length clamps to shield user from edges of glass plates
• Chemically tempered glass plates that significantly reduce glass plate breaking due to
overheating and routine heating and cooling
• No exposed metallic parts
• Pour spout in lower buffer chamber allows radioactive buffer to be easily and safely
poured for disposal
Important:
This apparatus meets I.E.C. 1010-1†safety standards. Sequi-Gen GT systems are safe to
use when operated in accordance with the instructions. This instrument should not be
modified in any way. Alteration of this instrument will:
• Void the manufacturer’s warranty
• Void the IEC1010-1 safety certification
• Create a potential safety hazard
† IEC1010-1 is an internationally accepted electrical safety standard for laboratory instruments.
1
Bio-Rad is not responsible for any injury or damage caused by the use of this instrument
for purposes other than those for which it is intended, or by modifications to the instrument
not performed by Bio-Rad or an authorized agent.
Power to the Sequi-Gen GT cell is supplied by an external DC power supply. This power
supply must be ground isolated in such a way that the DC voltage output floats with respect
to ground. The recommended power supply for this apparatus is the PowerPac 3000 power
supply. The maximum specified operating parameters for the Sequi-Gen GT cell are:
Maximum operating voltage – 3,000 VDC
Maximum operating power – 100 Watts
Electrical current to the Sequi-Gen GT cell enters the unit through the top and bottom
safety covers, providing a safety interlock to the user. Current flow to the cell is broken when
either safety cover is removed. Do not attempt to circumvent this safety interlock. Always
turn the power supply off while working with the sequencing cell when the safety covers are
not connected.
No user-serviceable parts are contained in this apparatus. To insure electrical safety, do
not attempt to service this apparatus.
Caution — Arcing
Arcing within an electrophoresis cell is represented by sparks, smoke, or charred surfaces
created when an electrical short has developed. Arcing can occur if the buffer level drops
below the recommended height, if there is buffer leakage, or if loose electrical connections
exists. If arcing is detected during electrophoresis, immediately remove the source of
electrical current (i.e., turn off the power supply).
Always use a power supply that is capable of detecting electrical conditions that may
cause accidental electrical shock or damage to the apparatus. The PowerPac 3000 power
supply contains safety features such as arc, no load, overload, rapid change in resistance, and
ground leak detection capabilities, that will reduce the chance of accidental electrical shock
and damage to the electrophoresis cell.
Before every use, inspect all plastic parts, glass plates, all electrical cables, jacks, and
receptors for loose connections, cracks, charring, or corrosion. Do not use any part that is
cracked, chipped, charred, or corroded. These parts may cause arcing. Contact your Bio-Rad
representative before using a part that may cause buffer leaking or arcing.
Warning:
Never allow the gel to exceed 60 ˚C. Excessive heat may crack the plates or cause the
silicon bond of the IPC to deteriorate.
Make sure that the upper and lower buffer chambers are filled with buffer during
electrophoresis. Do not allow the buffer level to drop below the level of the short glass plate
of the upper buffer chamber or below the bottom of the IPC assembly in the lower buffer
chamber at any time.
Certain solvents and cleaning agents should be avoided with this unit. Refer to Section 3.2
for compatible solvents, reagents and cleaning agents.
Definition of Symbols
Caution, risk of electrical shock Caution (refer to accompanying documents)
2
System Components
Each Sequi-Gen GT system comes with the components listed in Table 1.1. Check your
unit to be sure all items are present. Note any damage to the unit which may have occurred
during shipping. Notify Bio-Rad Laboratories if any items are missing or damaged.
Table 1.1. Sequi-Gen GT System Components
Item Quantity
GT Universal Base 1
Stabilizer Bar 1
GT Safety Covers
1
1
GT IPC, with bonded inner (short) glass plate
2
1
Outer (long) Glass Plate
2
1
GT Clamp Set (left and right clamp)
3
1
Precision Caster Base
1
1
Precision Caster Gasket
1
1
Precision Caster Syringe
4
1
Precision Caster Tubing, 60 cm 1
Precision Caster Luer Tapers 4
IPC Drain Port/Tubing Connector 1
Gel Temperature Indicator 1
Vinyl Spacers, 0.4 mm thick
3
2
Vinyl Sharkstooth Comb, 0.4 mm thick
5
1
Leveling Bubble 1
Instruction Manual 1
1 Parts come in 21 cm or 38 cm widths
2 GT IPC and Glass Plates are 21 x 40, 21 x 50, 38 x 30 cm or 38 x 50 cm sizes
3 GT Clamp Sets and Vinyl Spacers are either 30 cm, 40 cm or 50 cm lengths
4 Syringe sizes are 60 cc for 21 cm systems and 140 cc for 38 cm systems
5 Vinyl Sharkstooth combs are 24 well (25 teeth) for 21 cm units or 49 well (50 teeth) for 38 cm units
See Section 6 for information on accessories and replacement parts.
General Description
The Sequi-Gen GT DNA sequencing cell uses several innovative design features that are
especially useful for DNA/RNA sequencing or other nucleic acid separation applications.
Sequi-Gen GT DNA sequencing cell features and benefits include:
Features Benefits
Unique, horizontal, syringe injected gel Easy gel casting without tape, grease, and
casting method acrylamide spills and waste
Upper buffer chamber heat Provides uniform gel temperature that
distribution system prevents smiling
Permanently sealed upper buffer No gaskets or grease needed to provide leakchamber free electrophoresis
(continued on the next page)
3
Features Benefits
A universal base accepts all gel dimensions, Modular system allows different sized
including wide and narrow gel formats of gels to be used with the same lower buffer
various lengths chamber
Injection molded parts Provides years of rigorous use
Chemically tempered glass plates Resists cracking due to overheating
and rough handling
One-piece, lever-operated clamps Conveniently and easily slides onto gel
sandwich, and shields edges of glass plates
from operator contact
Molded chambers with pour spouts or Easy and safe radioactive buffer disposal
drain ports
Machined vinyl spacers and Uniform thickness of combs and spacers
sharkstooth combs reduces well-to-well leakage during
sample loading
Sequi-Gen GT Buffer Heat Dissipation
Uneven dissipation of the Joule heat produced by the gel during electrophoresis causes
electrophoresis artifacts. “Smiling” is a common artifact that develops when a gel sandwich
loses heat more efficiently at the edges than in the center. When a gel runs hotter in the
center, the electrical resistance decreases, and more current flows down the center. As the
current flow increases, the gel heats even more. Thus a positive feedback loop is set up which
results in the lanes near the center of the gel running hotter, and therefore faster, than the
lanes near the edges. Smiling can lead to ambiguity in reading the sequence.
The Sequi-Gen GT cell employs natural convection and conduction of the upper buffer
to distribute heat evenly. The problems of uneven heat dissipation are avoided. Complicated,
expensive thermostatic plates are not necessary.
A thin, transparent, upper buffer chamber, called an IPC (Integral Plate Chamber), acts as
a heat sink across the full area of the gel. Convection occurs any time a slight temperature
gradient develops, mixing the buffer (and heat) to prevent smile patterns from developing.
Convection is the most effective way to distribute heat evenly. The upper buffer dampens
temperature fluctuations in the gel, and adds to the reproducibility of each run. The contact
between the buffer and the gel plate is direct and uniform. Thermal and physical stresses are
reduced. The sample loading wells are always at the same temperature as the gel, resulting in
fewer re-annealing problems. Bubbles of gas, generated by electrolysis along the cathode,
rise through the buffer. These bubbles also help to prevent temperature gradients from
forming by stirring the upper buffer while rising to the top of the IPC chamber.
Sequi-Gen GT Gel Casting
Because of their large size, casting sequencing gels has traditionally been extremely
problematic. Taping the bottom or sides of the glass plate sandwich is time consuming and
does not always result in a perfect seal. Thus, vacuum grease is required to seal corners and
edges. The user must then “wrestle” with the gel mold in order to pour the gel correctly.
Sliding glass plates, or plate dropping methods always result in acrylamide spills and waste.
Cleaning the hazardous neurotoxin after the spills is also time consuming.
4
The precision caster allows quick and easy gel casting without acrylamide spills or waste.
By casting the gel with a syringe through the precision caster base, gels can be poured in less
than 1 minute. The gel is cast with the glass plate assembly in the horizontal position. Two
full-length clamps secure the assembly and allow attachment of the precision caster base to
the bottom of the glass plate sandwich. A seal between the caster gasket and the plates is
created without tape or grease. The gel is injected from the bottom of the glass plate sandwich
(via the injection port of the precision caster base) and moves to the top of the glass plates as
a dome-shaped gel front. Acrylamide spills and waste can be eliminated by controlling the flow
of the gel front at the top of the glass plates.
Modular Assembly
There are four IPC dimensions to choose from, as shown in Figure 1.1. One universal
base functions as the lower buffer chamber for all IPC sizes.
Fig. 1.1. Interchangeable sizes.
1.2 Specifications
General Specifications
Base footprint 16 x 48 cm
Maximum unit height 65 cm (50 cm cells); 55 cm (40 cm cells); 45 cm (30 cm cells)
IPC sizes 21 x 40, 21 x 50, 38 x 30 cm and 38 x 50 cm
(width x length)
Actual gel sizes 17 x 40, 17 x 50, 34 x 30 cm, 34 x 50 cm
Gel thickness range 0.25 – 0.75 mm
Nominal gel volumes (0.25 mm) 17 ml (21 x 40 cm); 21 ml (21 x 50 cm); 40 ml (38 x 30 cm);
43 ml (38 x 50 cm)
Nominal gel volumes (0.40 mm) 27 ml (21 x 40 cm); 34 ml (21 x 50 cm); 50 ml (38 x 30 cm);
68 ml (38 x 50 cm)
Minimum upper buffer volumes 500 ml (21 x 40 cm); 575 ml (21 x 50 cm); 650 ml
(38 x 30 cm); 1,400 ml (38 x 50 cm)
Minimum lower buffer volume 350 ml
Maximum lower buffer volume 500 ml
Electrical Specifications
Electrical Safety Certification IEC 1010-1
Rated voltage limit 3,000 volts
Rated power limit 100 watts
Rated temperature limit 60 ˚C
Electrical cables Rated to 3,000 volts (VDC)
Electrical leads Rated to 3,000 volts (VDC)
Banana plugs Rated to 3,000 volts (VDC)
5
21 x 40 cm 21 x 50 cm 38 x 30 cm 38 x 50 cm
Construction Specifications
GT IPC panel Injected molded polycarbonate
GT safety covers Injected molded polycarbonate
Universal base Injected molded polycarbonate
Stabilizer bar Injected molded polycarbonate
GT clamp set PVC clamp body
Protruded G10 polyester/glass cam shaft
Polycarbonate insulated stainless steel rod
Glass plates Chemically tempered 4.8 mm float glass
Combs and spacers Plastic or machined vinyl (see Sections 2.7 and 6.1)
Electrodes (IPC and base) Platinum, 0.25 mm diameter
Banana plugs (IPC and base) Gold plated stainless steel, 5.08 cm length
Electrical cables Dual, 20 AWG, tinned copper wire cable
Flame retardant polyurethane insulation jacket
Electrical leads Polyurethane insulated nickel silver, 2.95 cm length
Precision caster base Injection Molded Polycarbonate
Tubing Polyurethane, 3.2 mm internal diameter,
4.8 mm outer diameter
Luer taper Polypropylene, 3.2 mm internal diameter
Gasket Silicon Foam Sponge
Syringe Polypropylene, 60 cc or 140 cc
Drain port connector Polypropylene (quick coupling assembly)
3.2 mm internal flow diameter
Section 2
Description of Major Parts
2.1 Sequi-Gen GT Parts
See Figures 2.1 and 2.2 for Sequi-Gen GT part identification.
Fig. 2.1. Sequi-Gen GT gel casting parts.
6
Syringe
Tubing
Luer Taper
Precision Caster Base
Syringe
Sharkstooth Comb
Leveling Bubble
Drain Port
Precision Caster Cam Peg
Precision Caster Base Injection Port
GT Lever Clamps
Fig. 2.2. Sequi-Gen GT nucleic acid electrophoresis cell.
2.2 Gel Reagents and Electrophoresis Buffers
For most DNA sequencing or nucleic acid separations, a 19:1 acrylamide:bis solution is
required. A 1x TBE (Tris, boric acid and EDTA) solution is the preferred electrophoresis
buffer. Reproducibility is affected by the quality of the gel and buffer reagents. A full line of
high quality polyacrylamide gel reagents and nucleic acid electrophoresis buffers is available
from Bio-Rad. Premixed reagents and buffers are also available and offer convenience, time
savings, and reproducible results. Each reagent and buffer is purified to meet rigorous quality
control standards. See Section 6.2 for ordering information.
Sequi-Gen
®
GT
Sequencing Cell
7
GT Base
Leveling Feet
GT Bottom
Safety Cover
Stabilizer
Bar
IPC
(Integral
Plate
Chamber)
GT Lever
Clamp
GT Lever
Clamp
GT Top
Safety
Cover
2.3 Electrical Path
Both electrode wires are positioned near the bottom of the gel. The upper buffer carries the
current from the cathode up to the top of the plates near the fill spout, where the gel is exposed.
The lower buffer contacts the gel at the bottom edge of the plates in the standard fashion
(See Figure 2.3).
Fig. 2.3. Electrical path through IPC (Integral Plate Chamber) to lower buffer reservoir.
Section 3
Cleaning and Maintenance
3.1 Cleaning and Siliconizing Plates
Important:
To insure “bubble-free” gels using the Sequi-Gen GT precision caster, the glass plates
must be thoroughly cleaned and the outer (long) glass plate siliconized or coated before
each use.
1. Clean both Sequi-Gen GT glass plates (IPC and outer plates) thoroughly before each use.
• Carefully place the plate into the sink and rinse with warm water.
• Pour powdered lab detergent (Alconox [Alconox, Inc.] or Micro [International
Products]) into a gloved hand and add sufficient water to make a paste.
• Apply the paste and scrub the entire glass surface with a gloved hand, using circular
motions.
8
Fill spout
Upper buffer
Polycarbonate
panel
Bonded glass
plate
Gel
Outer glass
plate
Silicone
adhesive bond
IPC
drain port
Lower
buffer
• Rinse off all of the detergent with warm water.
• Rinse with deionized water.
• Wipe the cleaned plate with a large lint free tissue to dry.
2. Inspect the plates carefully for pieces of detergent, dried polyacrylamide, or other particles.
Rewash if necessary.
3. Perform siliconizing under a fume hood, to reduce the hazard from breathing silanizing
reagent. Alternatively, several non-toxic, non-corrosive glass plate coating solutions are
commercially available. We recommend siliconizing or coating only the outer (long)
plate, so that when the plates are separated, the gel sticks to the IPC-bound glass plate.
• Use a glass Pasteur pipette to dispense 2 ml of the silanizing reagent onto the front
plate. Coat the plate completely and evenly by spreading the silanizing reagent on the
plate surface with a large lint free tissue, using a motion that travels from the top to the
bottom of the plate.
Caution: Do not siliconize the IPC plate unless hexane, heptane, or water is used as a
solvent in the silanizing reagent. Other organic solvents will craze or damage the IPC
plastic and weaken the adhesive bond.
• Never heat an IPC in an oven. Severe damage will result to the adhesive bond. Use
siliconizing compounds that react, or cure, at room temperature.
Note: If the gels will be fixed or stained, the IPC (short) plate should be siliconized or
coated, since its immersion into fixing or staining solutions is not recommended.
4. Prior to assembling the plates, apply a small amount of ethanol to each plate and rub to
dryness with a tissue. Using the same tissue, clean the spacers.
3.2 Cleaning Sequi-Gen GT Components
1. Rinse the universal base buffer chamber, stabilizer bar, combs, spacers and precision
caster base, gasket, syringe and tubing assembly with a mild detergent solution in warm
water. Use a soft-bristled brush or sponge to remove polyacrylamide gel pieces.
Note: Do not snag or break the electrode wire in the universal base while cleaning.
2. Rinse thoroughly with warm water and air dry.
Compatible Cleaning Agents for Polycarbonate Parts
Chemically compatible cleaners must be used to ensure long life of parts. These include:
• Aqueous solutions of soaps and mild detergents
• Organic solvents:
• Hexane
• Aliphatic hydrocarbons
• Alcohols
• Methanol
• Ethanol
• Isopropyl alcohol
• Dilute acids
9
Loading...