GS Gene Linker®

UV Chamber

Instruction
Manual

For Technical Service

Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
 Warranty Statement
Bio-Rad Laboratories GS Gene Linker chamber is warranted against defects in materials
and workmanship for 1 year. If any defects occur in the instrument during this warranty
period, Bio-Rad will repair or replace the defective parts free of charge. The following
defects, however, are specifically excluded:
1. Defects caused by improper operation.
2. Damage caused by accident or misuse.
3. Damage caused by disaster.
4. Corrosion due to use of improper solvents or detergents.
5. Repair or modification performed by anyone other than Bio-Rad Laboratories or
authorized agent.
6. Use of spare parts supplied by anyone other than Bio-Rad Laboratories or authorized
agent.
The following components are not covered under warranty:
1. Fuses
2. Bulbs
Note: There are no user-serviceable parts or components inside the unit.
Unauthorized service or repair may void the warranty.
For any inquiry regarding instrument operation, call Bio-Rad Technical Service at
1-(800) 4BIORAD, or contact your local representative.
For any request for repair service, determine the model number and serial number of
your instrument, the purchase order number and invoice number, and in the U.S. call
Bio-Rad Instrument Service at 1-(800) 876-7614.
 Table of Contents

Section 1 Introduction .......................................................................................................1

Section 2 Specifications .....................................................................................................1
2.1 Safety ....................................................................................................................1
2.2 Specifications ........................................................................................................2
2.3 Components ..........................................................................................................3
Section 3 Operating Instructions....................................................................................4
3.1 Set Up....................................................................................................................4
3.2 Start/Stop...............................................................................................................4
3.3 Modes....................................................................................................................4
3.4 Monitoring ............................................................................................................5
3.5 Quick Reference Chart..........................................................................................6
Section 4 Methods ..............................................................................................................7
4.1 DNA Crosslinking.................................................................................................7
4.2 Sterilization ...........................................................................................................8
4.3 DNA Nicking ........................................................................................................8
Section 5 Cleaning and Maintenance ..........................................................................10
5.1 Cleaning ..............................................................................................................10
5.2 Bulb Replacement ...............................................................................................10
Section 6 Equipment and Accessories .........................................................................10
6.1 Gene Linker UV Chamber and Accessories .......................................................10
6.2 Electrophoresis Reagents and Equipment...........................................................10
6.3 Transfer Reagents and Equipment ......................................................................11
Section 7 References ........................................................................................................12
7.1 UV Crosslinking of DNA to Membrane .............................................................12
7.2 UV Crosslinking of DNA to Protein...................................................................12
7.3 UV Nicking of DNA ...........................................................................................13
7.4 UV Footprinting of DNA ....................................................................................13
7.5 Repair of UV-Induced DNA Damage.................................................................13
7.6 UV Crosslinking of RNA to Membrane .............................................................14
7.7 UV Crosslinking of RNA to Protein ...................................................................14
7.8 Cellular UV .........................................................................................................15
7.9 UV-Induced Mutagenesis ...................................................................................15
7.10 UV Sterilization of PCR Products ......................................................................15
Section 1
Introduction
The GS Gene Linker UV chamber is useful for all molecular biology applications
requiring UV irradiation. The GS Gene Linker chamber has three different operational
modes: Program, Energy, and Time. The Program mode has preset energy or time
parameters for specific applications. You can use one of these preset programs, or you
can set your own desired energy level or time using the Energy mode or the Time mode.
This manual provides information on how to operate the GS Gene Linker chamber along
with valuable application guidelines.

Section 2
Specifications
UV

2.1 Safety

Definition of Symbols
UV

Caution, risk of UV exposure Caution (refer to accompanying document)

Warning

This instrument is intended for laboratory use only.

This product conforms to the “Class A” standards for electromagnetic emissions for
laboratory equipment applications. It is possible that emissions from this product may
interfere with some sensitive appliances when placed nearby or in the same circuit as
those appliances. The user should be aware of this potential and take appropriate
measures to avoid interference.

UV

Lamps
The lamps inside this instrument emit short wave UV radiation. Overexposure to
direct or reflected UV light can cause severe damage to the eyes and skin. Never look
into an illuminated UV lamp without proper eye protection.
Ozone may be formed near UV lamps. Excessive ozone exposure can cause eye irri-
tation and discomfort in the respiratory tract. Operate the GS Gene Linker chamber in an
adequately ventilated area if ozone is detected by measurement or odor.

Sensor
The UV emission sensor is located on the right-hand inside wall. The UV sensor
detects only the energy output in the range of 200-400 nm, and will automatically turn
off the bulbs when the desired amount of cumulative UV energy has been delivered. The
UV sensor also measures maximum energy output from the bulbs, and the Replace Bulb
light will illuminate when the energy output is too low (approximately 30%). For these
reasons, the sensor should remain clear and clean for accurate energy readings (refer to
Section 5 for maintenance).

1
Safety Features
Power Switch The power switch can be used at any time to stop any of the operations.
Start/Stop The Start/Stop button will stop any of the on-going operations. If pressed
again the operation will start at the beginning of the cycle.
Open Door The GS Gene Linker chamber will not operate when the chamber door is
open. If the door is open, the Door Open light will be on. When the door
is properly closed the Door Open light will turn off. The chamber will
stop operating when the door is opened, but will resume operation when
the door is closed.
Chamber The sealed chamber protects you from any UV radiation. The viewing
window will protect your eyes and body from UV radiation.
Maximum The GS Gene Linker chamber is equipped with an automatic shut off after
Operation 999 seconds of energy emission if the detector is covered, or 24 minutes if
the radiometer check is not turned off.

2.2 Specifications

Dimensions Width x Depth x Height

Outside 42.7 x 30.5 x 26.4 cm (16.8 x 12 x 10.4 inches)
Inside 31.7 x 24.1 x 15.2 cm (12.5 x 9.5 x 6 inches)
Weight 10 kg (22 lb)

Functional
Input voltage range 100 VAC/50 Hz/1 amps
120 VAC/60 Hz/1 amps
220 VAC/50 Hz/0.5 amps
240 VAC/50 Hz/0.5 amps
Fuses 2.0 amp Slow-Blow (100/120 V) or
1.0 amp Slow-Blow (220/240 V) Type T
Environmental
Operating 50 ° F (10 °C) to 90 °F (32 °C) temperature
30–80% humidity
Storage 32 ° F (0 °C) to 140 °F (60 °C) temperature
10–90% humidity

UV Energy Source
Germicidal bulbs (5) G8T5 format, minibipin
Output 253.7 nm energy maximum

Note: This equipment has been tested and found to comply with the limits for a Class A
digital device, pursuant to Part 15 of the FCC rules. These limits provide reasonable
protection against harmful interference when the equipment is operated in a commercial
environment. This equipment generates, uses, and can radiate radio frequency energy
and, if not installed and used in accordance with the instruction manual, may cause
harmful interference in which case the user will be required to correct the interference
at his own expense.

2
2.3 Components
1. Power switch Power switch to turn the GS Gene Linker chamber On
or Off.
2. LED display Selected Program, Energy, or Time is displayed here.
3. Reference chart Quick reference on preset programs.
4. Program Program button is used to select the program mode.
5. Energy Energy button is used to select the energy mode. Energy is
measured in milliJoules.
6. Time Time button is used to select the time mode. Time is mea-
sured in seconds.
7. Actual energy Program light and Energy light are both on when the cumula-
tive energy is displayed on the LED.
8. Elapsed time Energy light and Time light are both on when the elapsed
time is displayed on the LED.
9. Start/Stop Start/Stop button is used to start the irradiation cycle or to
stop a cycle.
10. Raise Increases selected mode.
11. Lower Decreases selected mode.
12. Handle To open and close chamber door.
13. Door light Door Open light indicates the chamber door is open.
14. Bulb light Replace Bulb light indicates the UV bulbs need replacement.
15. Run light Run Complete light is on when the irradiation cycle has
ended.
16. Window Viewing window

3
Section 3
Operating Instructions
3.1 Set Up
1. Plug the chamber power cord into the appropriate electrical outlet.
2. Press the power switch On. The digital display should be C-L. If the display shows
ERR, the chamber is malfunctioning and needs repair. Contact your local Bio-Rad
representative, or call Bio-Rad Instrument Service at 1-800 876-7614.

3.2 Start/Stop
The Start/Stop button is used to start a given irradiation cycle. When a cycle is started,
the Start/Stop button can be used to stop the cycle. The cycle parameters will remain in
the temporary memory and can be restarted at the beginning of the cycle by pressing the
Start/Stop button again.
The light above the Start/Stop button will turn on when in use and turn off after the
irradiation cycle has ended.

3.3 Modes
The GS Gene Linker chamber can be operated in 3 different modes: Program, Time,
or Energy. The mode is indicated by the light above these buttons. The following instruc-
tions describe how to set and use each of these modes.

Program
Each Program is set in either energy or time. To see the preset program value, press
the Energy button to display the energy for this program, or press the Time button to dis-
play the time for this program. The LED will display - - - when the Time or Energy
parameter is not preset. Pressing the Raise or Lower buttons while viewing the set Time
or Energy switches out of the Program mode and into the indicated mode.
1. Place the material to be irradiated inside the GS Gene Linker chamber. Use filter
paper or plastic wrap to support membranes. Close the chamber door.
2. Press the Program button. Select the desired program by pressing the Raise or Lower
button.
3. Start the selected program by pressing the Start/Stop button. The light above the
Start/Stop button will be on during the irradiation. The LED will display Actual
Energy or Elapsed Time with the two lights above the buttons indicating which units
are being displayed.
4. The GS Gene Linker unit will automatically stop after reaching the set time or the set
energy level. At the end of the program, the GS Gene Linker chamber will sound a
tone for a few seconds. This tone can be stopped by pressing any button. The light
above the Start/Stop button will be off and the Run Complete light will be on. The
LED will display either energy or time indicated by the light above the button.

4
Energy
1. Place the material to be irradiated inside the GS Gene Linker chamber. Close the
chamber door.
2. Press the Energy button. Select the desired energy level by pressing the Raise or
Lower button.
3. Start the selected energy level by pressing the Start/Stop button. The light above the
Start/Stop button will be on during the irradiation. The LED will display the Actual
Energy with the light above the Program and Energy buttons on.
4. The GS Gene Linker unit will automatically shut down after reaching the set energy
level. At the end of the irradiation cycle, the GS Gene Linker chamber will sound a
tone for a few seconds. This tone can be stopped by pressing any button. The light
above the Start/Stop button will be off and the Run Complete light will be on. The
LED will display cumulative energy.

Time
1. Place the material to be irradiated inside the GS Gene Linker chamber. Close the
chamber door.
2. Press the Time button. Select the desired irradiation time by pressing the Raise or
Lower button.
3. Start the selected time by pressing the Start/Stop button. The light above the
Start/Stop button will be on during the irradiation. The LED will display the Elapsed
Time and the light above the Time and Energy buttons will be on.
4. The GS Gene Linker unit will automatically shut down after reaching the set time. At
the end of the irradiation cycle the GS Gene Linker chamber will sound a tone for a
few seconds. The tone can be stopped by pressing any button. The light above the
Start/Stop button will be off and the Run Complete LED will be on. The LED will
display elapsed time.

3.4 Monitoring
Any time during or after an irradiation cycle, the elapsed time or cumulative energy
can be determined.
1. To determine the elapsed time, press both the Energy button and the Time button
simultaneously. The elapsed time in seconds will appear on the LED.
2. To determine the cumulative energy, press both the Energy button and the Program
button simultaneously. The cumulative energy in milliJoules will appear on the LED.
3. Press the Energy or Time button alone and the LED will display the set values.

5
3.5 Quick Reference Chart
The GS Gene Linker chamber quick reference chart reproduced below, is located
behind a plexiglass shield on the chamber door. This chart can be used as an applications
guide. Bio-Rad will publish updated charts as new programs or protocols are developed.
To receive these updates, it is important for you to be listed in the GS Gene Linker cus-
tomer data base. Contact your Bio-Rad representative to be listed.

GS Gene Linker UV Chamber Quick Reference Chart

Make sure material is in the chamber and the door is properly closed. Turn ON
power. The LED display should read C-L. Select operation mode by pressing the
Program, Energy, or Time button. Press the Raise or Lower button to set the desired
program, energy level or time. Press the Start/Stop button to start the irradiation
cycle. At the completion of the cycle, a tone will sound and the UV light will auto-
matically turn off.

Program*
Application Conditions (LED Reading) Setting
Crosslinking Dot blot/NaOH NH4OAc C-L 125 mJoule
dry Zeta-Probe
Nicking Pulsed field gels nic 60 mJoule
Sterilization UV resistant material Str 90 sec
Crosslinking Dot blot/damp Zeta-Probe C1 30 mJoule
Crosslinking Southern dry membrane C2 50 mJoule
Crosslinking Southern damp membrane C3 150 mJoule
Crosslinking Dot blot/NaOH NH4OAc C4 250 mJoule
dry membrane 312 nm

*Program
(LED Reading) Section
C-L 4.1
nic 4.2
Str 4.1
C1 4.1
C2 4.3
C3 4.3
C4 [312 nm bulbs (to be released)]

6
Section 4
Methods
Bio-Rad Laboratories has developed protocols for UV crosslinking of nucleic acids
to nylon membrane, UV-induced nicking of DNA prior to transfer to membrane, and UV
sterilization. We have an ongoing research program to develop new protocols for molec-
ular biology applications. Our studies indicate that the energy required for optimal
crosslinking of nucleic acid to membrane is dependent upon several parameters. These
include the type of membrane (charged vs. neutral nylon), the transfer buffer, whether
the membrane is wet or dry, as well as the application (dot blot vs. genomic Southern).
The following protocols are recommended, depending upon the application.

4.1 DNA Crosslinking

Slot or Dot Blot/damp Zeta-Probe membrane
1. Denature the DNA in 0.4 N NaOH final concentration.
2. Assemble the microfiltration apparatus according to the manufacturer’s recommen-
dations. Use a positively charged nylon membrane like Zeta-Probe® GT membrane.
3. Apply the DNA samples directly onto the membrane.
4. Remove the membrane from the apparatus and place the damp membrane inside the
GS Gene Linker chamber. Use filter paper or plastic wrap to support the membrane.
5. Follow the instructions in Section 3.3, Program mode. Select the program with the
LED reading C1 (30 mJ). Start the irradiation cycle by pressing the Start/Stop but-
ton. Proceed with preferred hybridization protocol.

Slot or Dot Blot/NaOH NH4OAc/dry Zeta-Probe Membrane

1. Denature the DNA sample by adding NaOH and EDTA to a final concentration of
0.4 N NaOH and 10 mM EDTA. Heat the sample to 100 °C for 10 minutes.
2. Neutralize the DNA sample by adding an equal volume of cold 2 M ammonium
acetate (NH4OAc), pH 7.0.
3. Assemble the microfiltration apparatus according to the manufacturer’s recommen-
dations. Use a positively charged nylon membrane like Zeta-Probe GT membrane.
4. Apply the DNA samples directly onto the membrane.
5. Remove the membrane from the apparatus and place the damp membrane between 2
filter papers. Allow the membrane to dry. Remove the top filter paper and place
membrane inside the GS Gene Linker chamber.
6. Follow the instructions in Section 3.3, Program mode. Select the program with the
LED reading C-L (125 mJ). Start the irradiation cycle by pressing the Start/Stop but-
ton. Proceed with preferred hybridization protocol.
Note: When using a neutral membrane select program C3 (150 mJ).

Southern Transfer
1. Depurinate the DNA by soaking the gel in 0.25 N HCl for 10-15 minutes.
2. Denature the DNA by placing the gel in a bath of 0.5 N NaOH for 30 minutes.
3. Neutralize the gel by soaking it in 0.5 M Tris-HCl, pH 7.4, 1 M NaCl for 30 minutes.

7
4. Transfer the DNA onto a nylon membrane using 10x SSC or 10x SSPE as the trans-
fer buffer. The membrane can be a neutral nylon or a positively charged nylon mem-
brane like Zeta-Probe GT membrane.
5. After transfer, rinse the membrane in 2x SSC for 5 minutes. The membrane can be
damp or dry.
6. Follow the instructions in Section 3.3 Program mode. Select the program with the
LED reading C2 (50 mJ) if the membrane is dry, or C3 (150 mJ) if the membrane is
damp. Start the irradiation cycle by pressing the Start/Stop button. Proceed with pre-
ferred hybridization protocol.

4.2 Sterilization
The GS Gene Linker chamber has a sterilization cycle preset. Follow the instructions
in Section 3.3 Program mode. Select the third program with the LED reading Str (90 sec).
Start the irradiation cycle by pressing the Start/Stop button. The GS Gene Linker chamber
will sterilize only the exposed areas on any object in the chamber.

4.3 DNA Nicking25

The transfer efficiency of large DNA (>20 kb) from agarose gels is poor unless the
DNA is nicked prior to transfer to nylon membrane. The DNA can be cleaved either by
HCl depurination or by UV-irradiation. The depurination reaction is harder to control and
is extremely sensitive to temperature. Exposure to short wavelength UV light (254 nm) is
a more reliable method for nicking DNA in pulsed field gels before transfer. For optimal
results, the following protocol must be followed rigorously.
1. Stain the gel with 1.0 µg/ml ethidium bromide (EtBr) for exactly 30 minutes with
constant agitation. Do not destain the gel prior to nicking. Use a fresh dilution of
EtBr for each gel.
2. Place the gel on a glass tray for transporting the gel to the GS Gene Linker chamber.
3. Place the gel inside the chamber and close the door.
4. Follow the instructions in Section 3.3 Program mode. Select the second program
with the LED reading nic (60 mJ).
5. Start the irradiation cycle by pressing the Start/Stop button. The gel can be pho-
tographed, but exposure to UV radiation must be minimized (< 10 seconds). The gel
can be destained prior to photography if desired.
6. Soak the gel in 0.4 N NaOH, 1.5 M NaCl for 15 minutes.
7. Transfer the DNA onto Zeta-Probe GT membrane using two liters of 0.4 N NaOH,
1.5 M NaCl as the transfer solvent.
8. Set up the capillary transfer as follows, from bottom to top:
A. Corning Pyrex glass dish (28 x 18 x 4 cm).
B. A plexiglass or plastic box for support, about 3 cm high and small enough
to fit in the glass dish (e.g., Eppendorf yellow pipette tip rack).
C. Glass Plate (16 x 20 cm).
D. Three sheets of blotting paper as wick (18 x 30 cm) (S&S, GB002).

8
 E. Agarose gel (well side down).
F. Zeta-Probe membrane cut to the same size as the gel and pre-wetted with dis-
tilled water.
G. Three sheets of blotting paper (18 x 15 cm) (S&S, GB002).
H. A stack of paper towels 10 cm thick.
9. Transfer the DNA for 24–48 hours.
10. Carefully remove the paper towel and blotting papers. Remove the membrane
together with the gel, turn over the membrane and gel, lay them gel side up, and
mark the location of the wells and the orientation marker on the top of the gel. The
position of the wells can be accurately marked on the membrane by using a fine
point permanent alcohol marker pen, cutting through the bottoms of the wells.
11. Neutralize the membrane in 0.5 M Tris, pH 7.0 (neutralization buffer) for 5 minutes
followed by rinsing briefly in 2x SSC. Transferred DNA can be visualized on the
membrane by placing the damp blot on a transilluminator.
12. Dry the membrane by blotting on 3MM or other adsorbent paper and proceed to
hybridization. UV crosslinking of the DNA to the membrane is not recommended
with this alkaline transfer method.

Discussion
1. The procedure is based on gels approximately 6 mm thick. If thicker gels are used,
the staining period may be prolonged to allow diffusion of EtBr into the middle of
the gels. DNA that is not stained with EtBr will not be nicked by the UV light and
thus will not be transferred from the gel.
2. Presoaking the gel in NaOH prior to transfer decreases background and increases
transfer efficiency.
3. Pulsed field gels can also be blotted onto membranes using 10x SSC as the transfer
buffer with standard alkaline denaturation followed by neutralization. Alkaline trans-
fer onto nylon membranes gives as good or better sensitivity as standard transfers
onto nitrocellulose filters. The alkaline procedure is much simpler and faster. In
addition, nylon membranes can be reused many more times than nitrocellulose fil-
ters. Some blots may be reused as many as twenty times.
4. DNA separated on the CHEF-DR® II or CHEF Mapper® system can also be vacuum
transferred onto nylon membrane in 4 hours using the Model 785 Vacuum Blotter
(165-5001, 120 V) and NaOH as the transfer buffer.
5. The DNA is transferred from the back of the gel (the side opposite the wells) onto
the membrane because irregularities in the surface of the gel frequently occur during
solidification of these high percentage gels (1%). These surface artifacts will inter-
fere with the transfer of the DNAs from the gel. Transfer from the other side of the
gel insures smooth surface contact between the gel and the membrane.
6. It is essential to neutralize the membrane after transfer to prevent changing the pH of
the hybridization buffer during the hybridization.
7. It is not necessary to bake nylon membranes after alkaline transfer since the DNA
should be fixed onto the membrane by NaOH.

9
 8. To monitor the efficiency of the transfer, stain the gel in neutralization buffer for
30 minutes with 1.0 µg/ml EtBr. Photograph the post-transferred gel and compare
with the original picture.

Section 5
Cleaning and Maintenance
5.1 Cleaning
Outside - Clean the outside of the GS Gene Linker UV chamber with a damp towel.
Do not use solvents or strong detergents.
Inside - Clean the inside aluminum walls of the chamber with ethanol (reagent grade)
and a soft towel. The sensor is located on the right-hand inside wall. Clean the sensor
with a soft towel and ethanol. Be careful not to scratch the sensor with an abrasive towel.

5.2 Bulb Replacement

The GS Gene Linker chamber is equipped with a radiometer mode. This mode is
used to determine the bulb brightness. Enter into the radiometer mode by pressing both
the Raise and Lower buttons at the same time. The UV lights will turn on and the LED
will display an ‘L’ along with the UV illuminance measured in mW/cm2. After 1 minute,
the reading should stabilize. If the UV illuminance is ≤ 2 mW/cm2 ALL 5 bulbs should
be replaced. Stop the radiometer mode by pressing any button.
Turn off the GS Gene Linker chamber power and unplug the power cord. Remove
the bulb by holding either metal end and turn the bulb 1/4 turn. The bulb will slip out of
the socket through the slots on the bottom. Insert the new bulb through the slots and turn
the bulb 1/4 turn until the bulb snaps into place.

Section 6
Equipment and Accessories
6.1 GS Gene Linker UV Chamber and Accessories
165-5031 GS Gene Linker UV Chamber, 120 VAC/60 Hz; includes five 254 nm
bulbs and instruction manual
165-5032 GS Gene Linker UV Chamber, 220 VAC/50 Hz; includes five 254 nm
bulbs and instruction manual
165-5033 GS Gene Linker UV Chamber, 240 VAC/50 Hz; includes five 254 nm
bulbs and instruction manual
165-5034 GS Gene Linker UV Chamber, 100 VAC/50 Hz; includes five 254 nm
bulbs and instruction manual
165-5035 GS Gene Linker UV Chamber Replacement Bulbs, 254 nm, 5 bulbs

6.2 Electrophoresis Reagents and Equipment

170-4300 Sub-Cell DNA Electrophoresis Cell without Casting Tray
170-4304 Sub-Cell DNA Electrophoresis Cell with 15x20 cm Casting Tray
170-4343 Wide Mini-Sub Cell DNA Electrophoresis Cell Basic Unit
170-4307 Mini-Sub Cell DNA Electrophoresis Cell Basic Unit

10
Electrophoresis Reagents and Equipment (Continued)
165-5052 PowerPac 200 Power Supply, 100/120V
165-5053 PowerPac 200 Power Supply, 220/240V
Ultra Pure DNA Agaroses
162-0017 Low Melt Preparative Grade Agarose, 25 gm
162-0020 Low Melt Preparative Grade Agarose, 250 gm
162-0125 High Strength Analytical Grade Agarose, 100 gm
162-0126 High Strength Analytical Grade Agarose, 500 gm
162-0133 Molecular Biology Certified Agarose, 100 gm
162-0134 Molecular Biology Certified Agarose, 500 gm
162-0135 Chromosomal Grade Agarose, 25 gm
162-0136 Chromosomal Grade Agarose, 100 gm
161-0733 Premix 10x TBE Buffer, 1 liter

6.3 Transfer Reagents and Equipment

165-5000 Model 785 Vacuum Blotter with Regulator, includes: Vacuum
Regulator; Base unit with Vacuum Stage; Porous Vacuum Plate;
Reservoir Seal O-ring; Sealing Frame; Assortment Window Gaskets;
Vacuum Blotter lid; Instruction manual
165-5001 Model 785 Vacuum Blotter System: same as 165-5000 except with
vacuum pump (120 V)
165-5002 Model 785 Vacuum Blotter System: same as 165-5000 except with
vacuum pump (220/240 V)
165-5003 Model 785 Vacuum Blotter Basic Unit
170-6545 Bio-Dot Apparatus
170-6542 Bio-Dot Slot Format
170-3910 Trans-Blot Electrophoretic Transfer Cell
162-0190 Zeta-Probe GT Membrane, sheets, 9 x 12 cm, 15
162-0191 Zeta-Probe GT Membrane, sheets, 10 x 15 cm, 15
162-0192 Zeta-Probe GT Membrane, sheets, 15 x 15 cm, 15
162-0193 Zeta-Probe GT Membrane, sheets, 15 x 20 cm, 15
162-0194 Zeta-Probe GT Membrane, sheets, 20 x 20 cm, 15
162-0195 Zeta-Probe GT Membrane, sheets, 20 x 25 cm, 3
162-0196 Zeta-Probe GT Membrane, roll 30 cm x 3.3 m, 1
162-0197 Zeta-Probe GT Membrane, roll 20 cm x 3.3 m, 1
162-0198 Zeta-Probe GT Membrane, roll 30 cm x 30 m, 1
170-3557 Random Primer DNA Labeling Kit, 25 reactions
165-0962 Filter Paper Backing, 35 x 45 cm, 25 sheets
165-0921 Filter Paper Backing, 18 x 34 cm, 25 sheets

11
Section 7
References
7.1 UV Crosslinking of DNA to Membrane
1. Allefs, J.J.H.M.,Salentijn, E.M.J., Krens, F.A. and Rouwendal, G.J.A., Optimization of Non-
radioactive Southern Blot Hybridization: Single Copy Detection and Reuse of Blots. Nucleic
Acids Research, 18, (10) 3099-3100. (1990).
2. Cannon, Gordon, Heinhorst, Sabine, and Weissbach, Arthur, Quantitative Molecular
Hybridization on Nylon Membranes. Anal. Biochem. 149, 229-237. (1985).
3. Church, George M. and Gilbert, Walter, Genomic Sequencing. Proc. Natl. Acad. Sci. 81,
1991-1995. (1984).
4. Khandjian, E.W., Optimized Hybridization of DNA Blotted and Fixed to Nitrocellulose and
Nylon Membranes. Bio/Tech., 5, 165-167 (1987).
5. Knight, Pamela., Nucleic Acid and Protein Blotting. Bio/Tech. 8, 166-167 (1990).
6. Nierzwicki-Bauer, Sandra A., Gebhardt, Joan S., Linkkila, Leslie and Walsh, Kieron, A
Comparison of UV Cross-linking and Vacuum Baking for Nucleic Acid Immobilization and
Retention. Bio/Tech. 9, 472-478 (1990).
7. Razin, S.V., Yarovaya, O.V. and Georgiev, G.P. Low Ionic Strength Extraction of Nuclease-
Treated Nuclei Destroys the Attachment of Transcriptionally Active DNA to the Nuclear
Skeleton. Nucleic Acids Research, 13, 7427-7444 (1985).
8. Twomey, Tara A. and Krawetz, S.A. Parameters Affecting Hybridization of Nucleic Acids
Blotted onto Nylon or Nitrocellulose Membranes. Bio/Tech 8, 478-481 (1990).
9. Saluz, Hanspeter and Jost, Jean-Pierre, Optimized Genomic Sequencing as a Tool for the
Study of Cytosine Methylation in the Regulatory Region of the Checken Vitellogenin II Gene.
Gene 42, 151-157 (1986).

7.2 UV Crosslinking of DNA to Protein

10. Molitor, J. A., Walker, W., Doerre, S., Ballard, D.W., and Greene, W.C., NF-kB: A Family of
Inducible and Differentially Expressed Enhancer-Binding Proteins in Human T Cells. Proc.
Natl. Acad. Sci., 87, 10028-10032 (1990).
11. Gilmour, David S., Deitz, Thomas J. and Elgin, Sara C.R, UV Cross-linking Identifies Four
Polypeptides That Require the TATA Box to Bind to the Drosphila hsp70 Promoter. Mol. and
Cell. Bio,. 10, 4233-4238 (1990).
12. Dooley, Steven, Welter, Cornelius, Theisinger, Brigit and Blin, Nikolaus, Generating Highly
Labeled Oligonucleotides for DNA-Protein Interaction. GATA, 7(5), 133-137 (1990).
13. Meffert, Rainer, Rathgeber, Gabriele, Schader, Hans-Jochen and Dose, Klaus, UV-Induced
Cross-linking of Tet Repressor to DNA Containing Tet Operator Sequences and 8-
Azidoadenines. Nucleic Acids Research, 18 (22), 6633-6636 (1990).
14. Becker, Michael M., Lesser, David, Kurpiewski, Michael, Baranger, Anne and Jen-Jacobson,
Linda, Ultraviolet Footprinting Accurately Maps Sequence-Specific Contacts and DNA
Kinking in the EcoRI Endonuclease - DNA Complex. Proc. Natl. Acad. Sci., 85, 6247-6251
(1988).
15. Schnapp, Andreas, Clos, Joachim, Hadelt, Wolfgang, Schreck, Ralf., Cvekl, Ales and
Grummt, Ingrid, Isolation and Functional Characterization of TIF-IB, a Factor that Confers
Promoter Specificity to Mouse RNA Polymerase I. Nucleic Acids Research, 18(6), 1385-1393
(1990).
16. Schneider, Harald R., Waldschmidt, Rainer and Seifart, Klaus H., Human Transcription Factor
IIIC Contains a Polypeptide of 55 kDA Specifically Binding to Pol III Genes. Nucleic Acids
Research, 18(16), 4743-4750 (1990).
17. Pfleiderer, Christa, Smid, Amke, Bartsch, Ingrid and Grummt, Ingrid, An Undecamer DNA
Sequence Directs Termination of Human Ribosomal Gene Transcription. Nucleic Acids
Research, 18(16), 4727-4736 (1990).

12
18. Kaufman, J.D., Valanda, G., Rodriguez, G., Bushar, G., Giri, C., Norcross, MA., Mol. Cell
Biol., 7, 3759-3766, (1987).
19. Nabel, G. and Baltimore, D., Nature, 326, 711-713, (1987).
20. Tong-Starksen, S.E., Luciw, P.C., Peterlin, B.M., Proc. Natl. Acad. Sci. USA, 84, 6845-6849,
(1987).
21. Rosen, C.A., Sodroski, J.G., Haseltine, W.A., Cell, 41, 813-823, (1985).
22. Spandidos, D.A., Yiagnisis, M., Pintzas, A., Anticancer Res,. 9, 383-386, (1989).
23. Meffert, R. and Dose, K., FEBS Letters, 239, 190-194 (1988).

7.3 UV Nicking of DNA

24. Harding, Fiona A., Cohen, Nicholas and Litman, Gary W., Immunoglobulin Heavy Chain
Gene Organization and Complexity in the Skate, Raja erinaces. Nucleic Acids Research 18(4),
1015-1020 (1990).
25. Personal communication with Eric Lai University of North Carolina, Chapel Hill.

7.4 UV Footprinting of DNA

26. Uchida, Kiyoshi, Pyle, Anna Marie, Morii, Takashi and Barton, Jacqueline K., High
Resolution Footprinting of EcoRI and Distamycin with Rh(phi) 2 (bpy) 3+ , a New
Photofootprinting Reagent. Nucleic Acids Research, 17(24), 10259-10279 (1989).

7.5 Repair of UV-Induced DNA Damage

27. Freeman, Ph.D., Steven, E., Gange, M.D., Richard W., Sutherland, PhD., John C., Matzinger,
B.A., Ezra A. and Sutherland, PhD., Betsy M., Production of Pyrimidine. Dimers in DNA of
Human Skin Exposed in Situ to UVA Radiation. The Journal for Investigative Dermatology,
88, 430-433 (1987).
28. Gekik, Catherine M. and Collins, Andrew R., Comparison of Effects of Fostriecin,
Novobiocin, and Camptothecin, Inhibitors of DNA Topoisomerases, on DNA Replication and
Repair in Human Cells. Nucleic Acids Research, 18(4), 1007-1013 (1990).
29. Lyamichev, Victor I., Frank-Kamenetskii, Maxim D. and Soyfer, Valery N., Protection
Against UV-Induced Pyrimidine Dimerization in DNA by Triplex Formation. Nature, 344,
568-570 (1990).
30. Smerdon , Michael J., Bedoyan, Jirair and Thoma, Fritz., DNA Repair in a Small Yeast
Plasmid Folded into Chromatin. Nucleic Acids Research, 18(8), 2045-2051 (1990).
31. Chu, Gilbert and Chang, Elaine., Cisplatin-Resistant Cells Express Increased Levels of a
Factor That Recognizes Damaged DNA. Natl. Acad. Sci., 87, 3324-3327 (1990).
32. Freeman, Steven E. and Thompson, Bryan D., Quantitation of Ultraviolet Radiation-Induced
Cyclobutyl Pyrimidine Dimers in DNA by Video and Photographic Densitometry. Analytical
Biochemistry, 186, 222-228 (1990).
33. Tomkinson, Alan E., Bonk, R. Thomas., Kim, Joon., Bartfeld, Neil and Linn, Stuart.,
Mammalian Mitochondrial Endonuclease Activities Specific for Ultraviolet-Irradiated DNA.,
Nucleic Acids Research, 18(4), 929-935 (1989).
34. Madura, Kiran, Prakash, Satya and Prakash, Louise, Expression of the Saccharomyces cere-
visiae DNA Repair Gene RAD6 that Encodes a Ubiquitin Conjugating Enzyme, Increases in
Response to DNA Damage and in Meiosis but Remains Constant During the Mitotic Cell
Cycle, Nucleic Acids Research, 18(4), 771-778 (1990).
35. Tsujimura, Tohru, Maher, Veronica M., Godwin, Alan R., Liskay, Michael and McCormick, J.
Justin, Frequency of Intrachromosomal Homologous Recombination Induced by UV
Radiation in Normally Repairing and Excision Repair-Deficient Human Cells. Proc. Natl.
Acad. Sci., 87, 1566-1570 (1990).
36. Venema, J., Mullenders, L.H.F., Natarajan, A.T., van Zeeland, A.A. and Mayne, L.V., The
Genetic Defect in Cockayne Syndrome is Associated With a Defect in Repair of UV-Induced
DNA Damage in Transcriptionally Active DNA. Proc. Natl. Acad. Sci, 87, 4707-4711 (1990).

13
37. Madura, Kiran and Prakash, Satya, Transcript Levels of Saccharomyces cerevisiae DNA
Repair Gene RAD23 Increase in Response to UV Light and in Meiosis but Remain Constant
in the Mitotic Cell Cycle. Nucleic Acids Research, 18(16), 4737-4742 (1990).
38. Mitchell, David, L., Brash, Douglas E. and Nairn, Rodney S., Rapid Repair Kinetics of
Pyrimidine(6-4)Pyrimidine Photoproducts in Human Cells are Due to Excision Rather Than
Conformational Change. Nucleic Acids Research, 18(4), 963-971 (1990).
39. Ohnishi, Takeo, Yuba, Shunsuke, Date, Takayasu, Utsumi, Hiroshi and Matsukage, Akio, Rat
DNA Polymerase B Gene Can Join in Excision Repair of Escherichia coli. Nucleic Acids
Research, 18(19), 5673-5676 (1990).

7.6 UV Crosslinking of RNA to Membrane

40. Fingerle, J., Johnson, R., Clowes, A.W., Majesky, M.W. and Reidy, M.A. Role of uin Smooth
Muscle Cell Proliferation and Migration After Vascular Injury in Rat Carotid artery. Proc.
Natl. Acad Sci., 86, 8412-8416 (1989).
41. Herrin, D.L, and Schmidt, G.W., Rapid, Reversible Staining of Northern Blots Prior to
Hybridization. Bio/Tech, 6, 196-198 (1988).
42. Khandjian, Edouard W. and Meric, Claude A., Procedure for Northern Blot Analysis of Native
RNA. Anal. Biochem., 159, 227-232 (1986).
43. Kroczek, R.A., Mediate Visualization of Blotted RNA in Northern Analysis. Nucleic Acids
Research., 17, 9497 (1989).
44. Lin, Leu-Fen, H., Mismer, Drzislav, Lile, Jack D., Armes, Lyman G., Butler, Eugene T.,
Vannice, James L., Collins, Frank, Purification, Cloning and Expression of Ciliary
Neurotrophic Factor (CNTF). Science, 1023-1025 (1989).
45. Williams, Simon C., Bruckheimer, Sally M., Lusis, Aldons, J., LeBoeuf, R.C. and Kinniburgh,
Alan J., Mouse Apolipoprotein A-IV Gene: Nucleotide Sequence and Induction by a High-
Lipid Diet. Mol. Cell. Bio. 6, 3807-3814 (1986).
46. Kroczek, Richard A. and Siebert, Erwin, Optimization of Northern Analysis by Vacuum-
Blotting, RNA-Transfer Visualization and Ultraviolet Fixation. Analytical Biochemistry, 184,
90-95 (1990).
47. Farrell, Jr., Robert E., Methodologies for RNA Characterization II: Quantitation by Northern
Blot Analysis and the S1 Nuclease Assay. Clinical Biotechnology, 2(2), 107-119 (1990).

7.7 UV Crosslinking of RNA to Protein

48. Garcia-Blanco, Mariano A., Anderson, Gordon J., Beggs, Jean and Sharp, Phillip A., A
Mammalian Protein of 220 kDa Binds Pre-mRNAs in the Spliceosome: A Potential
Homologue of the Yeast PRP8 Protein. Proc. Natl. Acad. Sci., 87, 3082-3086 (1990).
49. Marciniak, Robert A., Garcia-Blanco, Mariano A. and Sharp, Phillip A., Identification and
Characterization of a HeLa Nuclear Protein that Specifically Binds to the Trans-Activation-
Response (TAR) Element of Human Immunodeficiency Virus. Proc. Natl. Acad. Sci., 87,
3624-3628 (1990).
50. Leibold, Elizabeth, A., Laudano, Andrew and Yu, Yang., Structural Requirements of Iron-
Responsive Elements for Binding of the Protein Involved in Both Transferring Receptor and
Ferritin mRNA Post-Transcriptional Regulation. Nucleic Acids Research, 18(7), 1819-1824
(1990).
51. Siebel, Christian W. and Rio, Donald C., Regulated Splicing of the Drosophilia P
Transposable Element Third Intron in Vitro: Somatic Repression. Science, 248, 1200-1208
(1990).

14
7.8 Cellular UV
52. Satokata, Ichiro, Tanaka, Kiyoji, Miura, Naoyuki, et al., Characterization of a Splicing
Mutation in Group A Xeroderma Pigmentosum. Proc. Natl. Acad. Sci., 87, 9908-9912 (1990).
53. Lawrence, C.W., Borden, A., Banerjee, S.K. and LeCler, J.C., Mutation Frequency and
Spectrum Resulting From a Single Abasic Site in a Single-Stranded Vector. Nucleic Acids
Research, 18(8), 2153-2157 (1990).

7.9 UV-Induced Mutagenesis

53. Armstrong, John D. and Kunz, Bernard A., Site and Strand Specificity of UVB Mutagenesis in
the SUP4-0 Gene of Yeast. Proc. Natl. Acad. Sci., 87, 9005-9009 (1990).

7.10 UV Sterilization of PCR Products

54. Rich, Jessica J., and Willis, David, A Single Oligonucleotide Can be Used to Rapidly Isolate
DNA Sequences Flanking a Transposon Tn5 Insertion by the Polymerase Chain Reaction,
Nucleic Acids Research, 18(22), 6673-6676 (1990).
55. Sarkar, Gobinda, and Sommer, Steve S., Parameters Affecting Susceptibility of PCR
Contamination to UV Inactivation, Biotechniques 10(5), 591-593 (1991).

The PCR process is covered by U.S. patent numbers 4,683,195, 4,683,202, and 4,899,818 which are owned by Hoffmann-
La Roche, Inc. and F. Hoffmann-La Roche, Ltd. The purchase of this product does not convey a license to use the process
covered by these patents. The user of this product to perform PCR must obtain a license from Hoffmann-La Roche, Inc.

15
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