UVP HybriCycler User Manual

HybriCycler Hybridization Oven

Operating Instructions

IMPORTANT: Please read these instructions before operating your UVP HybriCyclerTM

system to familiarize yourself with operation procedures.

81-0187-01 Rev C

HC-3000 HybriCycler Hybridization Oven 2

Introduction

The HybriCycler Hybridization Oven is designed as a compact alternative for
hybridization requirements. The HybriCycler Oven features microprocessor operation for
precise mixing capability. This space-saving design is manufactured with a 3/8” thick
beta-blocking acrylic and polycarbonate cylinder providing 360° view of the bottles. The
HybriCycler Oven housing is manufactured with a scratch-resistant, powder-painted
epoxy finish for easy cleaning and maintenance.

The HybriCycler operates at 11 RPM, enabling consistent saturation of samples,
whether is for washing or hybridizing. Having the ability to remove the carousel allows
for additional bottle size capacity. An easily removable protective tray under the rocker
wheel allows easy clean-up of spilled media.

A touch sensitive keypad and microprocessor control accurate temperature output from
ambient +10
desired hybridization temperature is entered, the readout displays the current
temperature inside the chamber. The HybriCycler Oven features:

o

C to 80oC. The keypad is located just below a large LED readout. Once the

Temperature Display

Power Controls

Carousel Chamber

Bottle Carousel

Spill Tray

HC-3000 HybriCycler Hybridization Oven 3

HybriCycler Hybridization Oven Hardware

The HybriCycler Oven is comprised of:

Hybridization Chamber

The space-saving Hybridization Chamber
is manufactured of 3/8” thick beta-blocking
acrylic providing a 360° view of the bottles.

Bottle Carousel

The cartridge-style bottle carousel is
manufactured of ¼” thick polycarbonate.
The carousel has the capacity to hold

• Four 35x300mm bottles

• Eight 35x150mm bottles

• Eight 50ml bottles

or one oversize bottle 70x300mm by
removing the carousel.

Spill Tray

The drawer-style spill tray is designed to
contain spills that leak from any of the
bottles within the Hybridization Chamber.
The spill tray has a fluid capacity of 300ml.

HC-3000 HybriCycler Hybridization Oven 4

Set-Up Instructions

This equipment is not intended for interconnection with any other devices. Use of this equipment
other than intended may create a safety hazard and/or malfunction.

1. Plug the female end of the power cord

to the connection on the back of the
HybriCycler.

2. Insert your Hybridization bottle(s) into

the bottle carousel. Start by sliding the
bottle into the bottle carousel’s entry
hole at either end of the bottle
carousel.

WARNING: Do not insert a bottle into the
center tube. To do so would block air flow
and may result in a safety hazard and or
malfunction.

3.

When the end of the hybridization
bottle is about to meet the bottle
carousel’s exit hole on the opposite
end, push the bottle down toward the
center of the bottle carousel, which
secures the bottle into the securing
rings. Then slide the hybridization
bottle through the bottle carousel’s exit
hole.

4. Open the HybriCycler’s door by

pressing down on the rear portion of
spring-loaded door latch. Slide the
bottle carousel into the hybridization
chamber ensuring that the front disk is
inserted into the groove of the carousel
guide. Close the door by squeezing the
door against the hybridization chamber
until the spring-loaded door latch
secures.

HC-3000 HybriCycler Hybridization Oven 5

Operation

HybriCycler functions:

• The “Power” switch turns the
HybriCycler ON or OFF.

• The “Motor” switch turns the bottle
rotation ON or OFF.

• The “Temperature Control” displays
and controls the temperature
setting within the hybridization
chamber.

1. To operate the unit, press the “Power”
switch to the ON position.

2. Set the “Temperature Control” to the
required temperature by pressing
either the “Up” or “Down” keypad arrow
buttons to raise or lower the displayed
degree (displayed in degrees Celcius).
This setting is adjustable from ambient

o

+10

C to 80.0°C. See Temperature
Control Instructions on page 6 for more
details.

HC-3000 HybriCycler Hybridization Oven 6

3. Press the “Motor” switch to the ON
position.

Setting the Temperature Controls

Whenever power is applied to the controller, the software revision number is displayed
for several seconds. While the software revision number is being displayed, the intensity
of the display digits alternates between full and half brightness, and on some models the
two outside decimals points blink on and off. After several seconds, the display reverts
to showing the controlled temperature. In this mode, at most a single decimal point is
illuminated, and the display intensity is steady.

Altering the Setpoint

The current setpoint value can be altered using the “UP” and “DOWN” buttons while the
setpoint is being displayed. To change the setpoint from normal mode, proceed as
follows:

• Momentarily press then release either the “UP” or the “DOWN” button. The LED
intensity will alternate between half and full intensity to indicate that the displayed
value is the current setpoint.

• Increase or decrease the setpoint value by pressing “UP” or “DOWN” buttons
respectively. If either button is held down for more than several seconds, setpoint
value will increase or decrease continuously.

• When the desired setpoint is reached, wait approximately five seconds without
pressing either button; the display will revert to normal mode showing the actual
temperature.

The new setpoint becomes effective and is stored in the non-volatile memory when the
display reverts to the normal mode.

Calibrating the Temperature Reading

The HybriCycler is calibrated at the UVP factory. UVP recommends temperature
recalibration be performed at the UVP factory during the warranty period as recalibration
by the user may void the warranty. If the user chooses to recalibrate the unit,
Temperature Calibration Reading instructions may be obtained from UVP by calling
UVP’s customer service department in Upland, California at [phone] or (909)
946-3197 or Cambridge, UK at [phone].

HC-3000 HybriCycler Hybridization Oven 7

Care and Cleaning

The drawer-style spill tray is designed to contain any spills that leak from any of the
bottles within the Hybridization Chamber. The spill tray has a fluid capacity of 300ml.
The inside of the HybriCycler Hybridization Chamber should be cleaned with a damp
cloth. The bottle carousel and outside of the HybriCycler can be cleaned with soap and
water. The chamber, rollers and spill tray may be decontaminated by wiping clean with a
decontaminating agent*, then repeat with distilled water. Rollers and carousel guide are
removable facilitate cleaning.

NOTE: *This decontamination method may not remove all contaminants. Refer to
Federal, State and Local Guidelines and Biological Protocols to assure decontamination.

If any unit requires service, a Returned Goods Authorization (RGA) must be obtained
from UVP’s customer service department prior to returning any equipment to UVP. If
radioactive or biological hazardous material has been used within the unit, radioactive
decontamination and biological clean-up as per current Federal, State and Local
Guidelines and Biological Protocols must be performed, BEFORE returning the unit to
UVP.

Replacement Parts

Replacement parts lists are provided below for the HC-3000 HybriCycler Systems.
Repairs or replacement other than specified in the following procedures shall be done
only by authorized service personnel.

Qty Part Number
Fuse, 10 Amp, 250V 5x20mm, Slo-Blo 2 ea 56-0002-04
Main Motor Switch 1 ea 53-0165-02
Power Cord, 115V 1 ea 58-0085-01
Power Cord, 230V 1 ea 58-0085-03
Adjustable Feet 4 ea 72-0062-01
Carousel Assembly 1 ea 76-0089-01
Roller Assembly 2 ea 76-0309-01

Bottle, Small 35x150mm 07-0194-02
Bottle, Large 35x300mm 07-0194-01

Technical Support and Maintenance

UVP offers technical support for all of its products. If you have any questions about the
product’s use, operation, or repair, please call or fax UVP Customer Service at the
following numbers:

In the US toll free [phone] or [phone], fax: [phone], email: [e-mail]
In Europe/UK call [phone] or fax: [phone], email: [e-mail]

HC-3000 HybriCycler Hybridization Oven 8

Hybridization Techniques

There are really two main steps to a hybridization reaction: hybridizing two strands of
complimentary DNA and detection of the hybridized DNA. Nucleic acid hybridization is a
mechanism where strands of DNA in a single stranded state have their complements
bind together. The proximity of the DNA strands to each other determines the frequency
of the binding events and is in fact successful binding is proportional to their
concentration. The concentration of the target (nucleic acid you are looking for) is the
independent variable in all hybridization reactions.
Since the target concentration is usually the unknown variable, an excess of labeled
probe (what you use to find the target) will drive the reaction, thus decreasing the time
for the probe to hybridize to a target. This is simply increasing the chances of a probe
bumping into a target. But with an enormous amount of probe around (in solution or on
the surface of a membrane) the background signal will also be enormous. The typical
approach to correct for excess background (noise) on a membrane or slide hybridization:
wash in a low salt buffer as this favors the disassociation of unbound probe from the
membrane/slide and non-complementary DNA. In solutions a probe can be
enzymatically degraded by using a single strand specific nuclease.

Mechanisms of Nucleic Hybridization

Hybridization occurs with a process called
nucleation whereby the two separate
nucleic acid strands come into close
proximity of each other. A duplex region is
formed where a minimum of three bases of
one strand complements to those on the
second strand. If the remainder of the
strands are complementary, the two
strands will anneal or zipper together very
quickly. The rate limiting step in nucleic
acid hybridization is the duplex formation,
which again explains why probe to target
concentrations are critical.

Experimental Protocols

There are many different protocols available on the web, in journals, and in text
references and we reference several at the end of this text.

HC-3000 HybriCycler Hybridization Oven 9

1: Concentration of Species

Target: How much target molecule depends on the species you expect to find.

Cellular constituents may be expressed in large or small amounts; the trick is to
start with enough target (~25 µg) and determine experimentally.

Probe: plan to have more probe than expected target. To answer questions
about adding too much probe; run an extinction experiment: serially increase the
amount of target by a factor of two and use a fixed amount of probe. Hybridize
for a short length of time and quantitate the amount of probe that has hybridized.
As long as the signal increases and shows linearity there is excess probe (Fig 2
left). If the signal levels off and a loss of linearity noticed, then the probe is not in
excess (Fig 2 right).

2: Length of Probe
The goal is to increase hybridization efficiency while minimizing background. In
most cases probes range from 20 – 1000 bps.

3: Salt Concentration and Temperature
Nucleic acid requires salt (monovalent cations) to reduce the ionic effects of the
phosphate backbone, and heat as a form of non-denaturing kinetic energy.
Because the salt concentration and temperature effect each other, knowing the
thermostability of the hybrid probe is helpful. Hybridization rate varies directly
with the sodium ion concentration between 0.03 and 1.2 M. Most protocols run
between 0.5 and 1.1 M Sodium.

HC-3000 HybriCycler Hybridization Oven 10

Situation Response
G+C = 45-55% follow normal protocol
G+C < 45% Lower salt and temperature
G+C > 55% Raise salt and temperature
Evidence of probe target

Lower salt and temperature

mismatching

Target and probe is

Hybridize in a formamide-based buffer
degraded on aqueous
solution

Unacceptable high

Use less probe
background

Hybridize at lower salt/ higher temperature

Wash with lower salt higher temperature

Incubate with very low salt/change nuclease(solution)

Use a smaller probe or a different probe

Clean probe of contaminants prior to use

4: Aqueous or Denaturing Hybridization Buffer

If hybridization takes place in an aqueous salt environment of 0.8 to 1.2M salt,
the T

(the temperature at which the half of the duplex molecules will dissociate

M½

under a given set of conditions) can be 90°C. This is high enough to degrade
DNA, RNA and some proteins. It is therefore possible to add formamide as a
denaturing / temperature lowering agent because for every percent of formamide
in the reaction the T

is reduced by 0.65°C. Therefore, at 80% formamide,

M½

reactions can be performed in the 40 - 55°C range. However the rate of
formamide-based hybridization is at least three-fold lower than that of aqueous
hybridization requiring longer incubations.

HC-3000 HybriCycler Hybridization Oven 11

Protocol 1: Random priming method for tagging DNA with fluorescein-lebeled
nucleotide and others

This method uses DNA polymerase to incorporate Fluorescene-11- dUTP into double
stranded DNA probes. This protocol can be used to incorporate any tagged nucleotides.

Equipment

 Micropipettes and tips

 Boiling water bath

 1.5 mL Microcentrifuge tubes

 Microcentrifuge

 Cap lock for Microcentrifuge tube

 Water bath set to 37°C

Reagents

 Deionized, sterile water

 EDTA, 0.5 M

 Klenow DNA polymerase , 4-5 units/µL

 Nucleotide mix (300µm each of dATP, dCTP, dGTP and 60 µm dTTP)

 Random nonamer (9-mer) primers, 2.5 µg/µL in water

 Reaction buffer, 10X: 50mM MgCl

mM Tris-HCl, pH 7.5

 Tagged nucleotide: fluorescene-11-dUTP

 Template DNA in water (5ng/mL)

Procedure

1. Pipette 10 mL of template DNA plus 10 mL of water into a
microcentrifuge tube and cap tightly. Cover cap with a cap lock or
bend a paper clip in half and secure over the microcentrifuge tube.

2. Place the tube into the boiling water bath for 5 minutes.

3. Immeadiately place tube on ice for 5 minutes.

4. Centrifuge for 15 seconds in microcentrifuge.

5. Add the reagents listed below to a fresh tube on ice in the following
order:

, 10mM 2-Mercaptoethanol, 500

2

6. 10 mL Nucleotide mix

7. 5 mL Tagged nucleotide

8. 5 mL Reaction buffer (x10)

9. 5 mL Random primers

10. 10 mL Boiled DNA

11. 14 mL Water

HC-3000 HybriCycler Hybridization Oven 12

12. 1 mL DNA polymerase

13. Mix gently and incubate at 37°C for 1 hour

14. Stop the reaction by adding 2 mL EDTA

15. Store probes at -20°C in the dark.

Protocol 2: Hybridization to Nylon or Nitrocellulose

Hybridization to nylon or nitrocellulose membranes containing Nucleic Acid is
accomplished by adding single-stranded probe to the membranes that have been
previously incubated with prehybridization solution. The prehybridization and
hybridization solutions both contain buffers designed to prevent adventitious binding of
the probe to the filters.

Reagents and Equipment

 Prehybridization/hybridization solution [45% formamide, 5X SSPE (0.9 M

NaCl, 50mM sodium phosphate buffer, pH 7.4, 5mM EDTA), 0.1% SDS,

5X Denhardt’s solution (0.1% each of Ficoll, polyvinylpyrrolidone, and

bovine serum albumin), and 100 mg/mL of denatured salmon sperm

DNA). Mix well and remove aggregates before use.

Notes: When preparing prehybridization/hybridization solutions, add dry
reagents directly to the formamide/SSC solution. Incubate with mixing at 40­50°C for 2 hours or until dissolved. Store at -20°C. SDS will precipitate at
room temperature but remain in solution at 37°C.

 UVP Hybridization bottle(s) and caps

 15 mL plastic tube

 Boiling Water Bath

 Bucket of ice

 Glove

 Plexiglass shield

 UVP Minidizer™, HybriCycler™, or Hybridizer™

Procedure

1. Add 15 mL of prehybridization solution to each hybridization bottle
containing the blot. Remove bubbles between the glass and blot.
Cap the blots and close the Hybridizer.

2. Incubate the blot at 42°C for 1 hour.

3. Remove prehybridization solution and replace with 10 mL of
hybridization solution.

4. Pipette 1x106 counts per minute of radio labeled probe or 200ng of
biotinylated DNA into a 15-mL plastic tube. Seal the tube with a
plastic cap and poke a hole in the top with a syringe needle to prevent
pressure build-up during boiling.

HC-3000 HybriCycler Hybridization Oven 13

5. Denature the probe by placing the samples in the boiling water bath
and heating for 10 minutes. Immediately transfer the tube to ice for 5
minutes (to prevent renaturation). Add 5 mL of hybridization buffer to
the probe and transfer to the hybridization bottle containing the blot:
AVOID pouring the probe directly onto the blot.

6. Incubate in the UVP HybriCycler, Hybridizer, or Minidizer 6 to 8 hours
at 42 to 56 degrees.

Washing the blot

 Tupperware container (sized to contain the blot)

 0.1X SSC, 0.1% SDS (pre-warmed to 50°C)

 2X SSC, 0.1 % SDS (room temperature)

 2X SSC (room temperature)

 0.15X SSC, 0.1% SDS (pre-warmed to 50°C)

 Gloves

 Filter Paper

 Cardboard

 Plastic wrap

 Tape

Non radioactive probes

1. Wash blots in 2X SSC, 0.1% SDS for 3 minutes at room temperature
(repeat one)

2. Wash filter in 0.15X SSC, 0.1%SDS for 15 minutes at 50°C (repeat once)

3. Store blots in 2X SSC at room temperature

Radioactive probes

Additionally you will need:

 X-Ray film holder

 X-Ray film

 Intensifying screen

Procedure

1. Remove blot from hybridization tube and transfer to Tupperware

container

2. Rinse briefly in 50°C 0.1X SSC, 0.1% SDS.

3. Remove this solution to radioactive waste and wash

4. Wash blot three more times in the same solution.

5. After the final wash, dry blot on filter paper for 10 minutes. This is a

good time to quickly pass your hand held radioisotope reader (beta or
gamma counter) over your blot to get a general idea as to the
exposure time you will need for the x-ray film. Hot blots are 20
minutes to 2 hours. Not so hot blots can be left overnight (8 hours).

HC-3000 HybriCycler Hybridization Oven 14

6. Tape the blot to a cardboard backing.

7. Cover with plastic wrap to prevent the blots from sticking to the x-ray

film.

8. Place the cardboard containing the blots into the X-ray film folder.

9. In the darkroom, place a piece of X-ray film over the blot(s).

10. Place an intensifying screen on top of the film.

11. Close the film folder and clamp it.

12. Store at –70°C. The low temperature reduces light scattering and

increases the length of exposure time. Expose the blot for 20 minutes
to 24 hours.

Protocol 3: Chemiluminescence detection: UVP BioWest ™, HRP­tagged, Alkaline phosphatase (AP) probes or antibody conjugates

Equipment

 Clear plastic cling-wrap or Clear transparent sheet protector

 UVP EpiChemi II™ Darkroom

 Pipette

 Cooled CCD camera

Reagents

UVP BioWest™ – For HRP Tagged Species

o BioWest Substrate Working Solution: Prepare by mixing equal

parts 1:1 BioWest Luminol/Enhancer Solution and BioWest Stable
Peroxide Solution. This working solution is stable for a minimum
of 24 hours at room temperature and may be stored in ambient
light and temperature.

BioWest Procedure

1. Immerse the blot in BioWest Substrate Working Solution for five

minutes. Completely immerse the blot in the substrate (0.125
mL/cm2).

2. Remove the blot from the substrate solution and place it in a sheet

protector.

3. Remove any bubbles between the blot and the surface of the

protector.

4. Place in an EpiChemi II Darkroom and with all internal lights off image

the blot.

Other Chemiluminescence

 ECL tm (or other) detection reagent 1

 ECL tm (or other if required) detection reagent 2

HC-3000 HybriCycler Hybridization Oven 15

Product Warranty

UVP’s quality HC-3000 HybriCycler System is guaranteed to be free of defects in
materials, workmanship and manufacture for one (1) year from the date of purchase.
Consumable and disposable parts including, but not limited to bottles, tubes and filters,
are guaranteed to be free from defects in manufacture and materials for ninety (90) days
from date of purchase. If equipment failure or malfunction occurs during the warranty
period, UVP shall examine the inoperative equipment and have the option of repairing or
replacing any part(s) which, in the judgment of UVP, were originally defective or became
so under conditions of normal usage and service.

No warranty shall apply to any instrument, or part thereof, that has been subject to
accident, negligence, alteration, abuse or misuse by the end-user. Moreover, UVP
makes no warranties whatsoever with respect to parts not supplied by UVP or that have
been installed, used and/or serviced other than in strict compliance with the instructions
appearing in the operational manual supplied to the end-user.

In no event shall UVP be responsible to the end-user for any incidental or consequential
damages, whether foreseeable or not, including, but not limited to property damage,
inability to use equipment, lost business, lost profits, or inconvenience arising out of or
connected with the use of instruments produced by UVP. Nor is UVP liable or
responsible for any personal injuries occurring as a result of the use, installation and/or
servicing of equipment. This warranty does not supersede any statutory rights that may
be available in certain countries.

NOTE: Slight visual aberrations in the acrylic are a natural occurrence during the
manufacturing/forming process and are not considered a defect.

UVP, Inc., 2066 W. 11th Street, Upland, California 91786

[phone] • [phone] Fax: [phone]

Ultra-Violet Products Limited, Unit 1, Trinity Hall Estate, Nuffield Rd. Cambridge, CB4 1TG UK

[phone] • Fax: [phone]

Web site: www.uvp.com

HybriCycler is a Trademark of UVP, Inc.