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

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.

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)

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

GT IPC, with bonded inner (short) glass plate

Outer (long) Glass Plate

GT Clamp Set (left and right clamp)

Precision Caster Base

Precision Caster Gasket

Precision Caster Syringe

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

2 Vinyl Sharkstooth Comb, 0.4 mm thick

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)

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.

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)

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.

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

Sequencing Cell

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.

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

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