Hybridization Ovens
Contents
1. Gloves Plexiglas shield UVP Minidizer or Hybridizer Hybridization Oven Procedure 1 6 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 Incubate the blot at 42 C for 1 hour Remove prehybridization solution and replace with 10 mL of hybridization solution 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 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 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 2 3 Page 21 Wash blots in 2X SSC 0 1 SDS for 3 minutes at room temperature repeat one Wash f2. The units are built to provide you with trouble free operation To ensure correct operation Wipe ANY water from inside and outside the unit with a soft cloth or sponge Use soap and water with a soft cloth or sponge to clean the unit Do not allow chemicals to remain on the unit s surfaces Never clean the unit with abrasive pads or cleaners Never clean the unit with acetone or chloroform Clean the UV sensor regularly with a soft cloth and alcohol Bottle Care Hybridization bottles are made of lead free borosilicate glass which provides superior long term reliability Each bottle includes a polypropylene cap and PTFE seal Page 13 It is important to check your bottles regularly for chips stress fractures and cracks If these occur the bottle must be discarded Ensure bottles are stored either in a suitable rack or with caps replaced in between experiments This will protect the bottle and sealing area Replace O rings PTFE seals when worn or leaky replace O rings or seals every six months Wear protective gloves to protect your hands in the event of an accidental breakage Never over tighten caps on bottles as this will harm the seals Hand tightening is sufficient If the cap is difficult to unscrew never attempt to force the bottle cap open Allow the bottle to cool and retry If the cap remains stuck discard the bottle The bottles should not be used at temperatures above 70 C 81 0169 01 Rev U Hybrid
3. 0085 01 58 0085 02 58 0085 03 PART NO 07 0194 02 88 0004 01 76 0070 01 76 0064 03 10 0292 01 34 0007 01 Contact UVP Contact UVP Contact UVP Contact UVP Contact UVP Contact UVP 53 0165 01 72 0030 01 72 0062 02 22 0110 01 53 0072 01 58 0085 01 58 0085 02 58 0085 03 81 0169 01 Rev U Hybridization Ovens Replacement Parts for HM 4000 Multidizer DESCRIPTION Borosilicate Glass Bottle with Cap and O Ring Large 35 x 300mm Cap and O Ring for all Bottle sizes Bottle Holder Carousel Carousel Drive Shaft Rod Drip Tray for Lower Chamber Hood Acrylic for Upper Chamber Roller Assembly Roller Insert and Cam Assembly Reciprocating Shaker Tray 10 W x 6 D Drip Tray for Upper Chamber Fuses Main Switch Knob Motor Control Adjustable Rubber Foot Door Handle Compression Latch Power Cord 115V Power Cord 230V QTY NEEDED PART NO 07 0194 02 88 0004 01 76 0070 01 76 0064 03 10 0292 02 N N 10 0317 01 76 0309 01 76 0309 02 98 0067 02 10 0318 01 a a Nl Contact UVP 53 0165 01 72 0030 01 72 0062 02 22 0110 01 ANA 58 0180 01 58 0180 02 Reciprocating Shaker Tray 81 0169 01 Rev U Page 15 Hybridization Ovens Accessories Bottles Borosilicate glass bottles with polypropylene cap and PTFE seal Bottle Small 35 x 150 mm 07 0194 01 Bottle Oversize Call UVP Bottle Carousels Carousel Kit for Four Oversized Bottles 76 0066 02 C
4. 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 a single decimal point is illuminated at most 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 7 Momentarily press then release either the UP or the DOWN button The LED display will flash to indicate that the displayed value is the current setpoint 8 Increase or decrease the setpoint value by pressing the UP or DOWN buttons respectively If either button is held down for more than several seconds the setpoint value will increase or decrease continuously 9 When the desired setpoint is reached wait approximately five seconds without pressing either button The display will then 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 Page 8 81 0169 01 Rev U Hybridization Ovens Calibrating the Temperature Reading The hybridizer is calibrated at the UVP factory UVP recommends temperature recalibration be performed at the UVP factory as recalibration by the user may void the warranty Obtain an RGA Returned Goods Authorization number
5. are many different protocols available on the web in journals and in text references and several are reference at the end of this text 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 ug 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 If the signal levels off and a loss of linearity noticed then the probe is not in excess Fig 2 2 Length of Probe The goal is to increase hybridization efficiency while minimizing background In most cases probes range from 20 1000 bps ss ay i a j m au MMM C 0 0 0 0 0 E F ke iz 2 o ol E E L a a i Fe E z a a Amount of target nucleic Amount cf target nucleic acid arbitrary units acid arbitrary units Figure 2 Analysis for probe excess in hybridization reactions Page 18 81 0169 01 Rev U Hybridization Ovens Page 19 3 Salt Concentration and Temperature Nucleic acid requires salt monovalent cations to red
6. from UVP prior to returning a unit to the factory Call UVP s customer service department in the USA in Upland California at 800 452 6788 or 909 946 3197 or in the UK in Cambridge UK at 44 0 1223420022 Decontamination IMPORTANT These decontamination methods may not remove all contaminants Refer to Federal State and Local Guidelines and Biological Protocols to assure decontamination Bottles and Caps e Soak items in a diluted detergent solution overnight e Remove from detergent and rinse items with distilled water e If items are still contaminated gently scrub with an abrasive cloth or brush If necessary continue to soak items in the detergent solution for a longer period of time Oven Chamber The oven chamber and drip pans may be decontaminated by wiping clean with a decontaminating agent then repeat with distilled water NOTE If any unit requires service a Returned Goods Authorization RGA number must be obtained from UVP s customer service department prior to returning any item to UVP If radioactive or biological hazardous material has been present within the unit radioactive decontamination and biological cleanup must be performed BEFORE returning the unit as per current federal state and local guidelines and biological protocols Using the Crosslinker The crosslinker portion of the HL 2000 HybriLinker operates independently from the hybridizer The crosslinker provides an exposure instrument which uti
7. 10 C to 99 9 C Temperature Stability Inside Oven 0 3 C to 68 C Inside Bottles 0 1 C to 68 C Temperature Uniformity Inside Oven 0 1 C to 68 C Inside Bottles 0 1 C to 68 C Temperature Display LCD Heating Element 1250 watts Rotation Speed Off 10 to 15 RPM Bottle Capacity Twenty 20 35 x 150 mm Ten 10 35 x 300 mm Interior Dimensions 14 W x 10 75 H x 10 75D 356 x 273 x 273mm Crosslinker Microprocessor controls 8 watt Shortwave UV 254nm tubes Large LED displaying time or energy Tactile membrane keypad Preset or user selected UV time exposure settings Internal interlocking safety system Preset or user selected UV energy settings Maximum UV energy setting 999 900 microjoules per cm Interior Dimensions 11 375W x 3 75H x 11 5D in 288 x 95 x 292 mm 81 0169 01 Rev U Hybridization Ovens HM 4000 Multidizer Hybridization Oven Page 5 Part Numbers Volts Hz 95 0340 01 115V 60Hz 95 0340 02 230V 50Hz 95 0340 03 100V 50Hz Specifications Net Weight 75 lbs 34 kg Temperature Display LCD Dimensions Exterior 17 5W x 28 5H x 15D in 445 x 724 x 381 mm Upper Chamber Temperature Ambient 10 C to 80 C Temperature Stability Inside Oven 0 3 C to 68 C Inside Bottles 0 1 C to 68 C Temperature Uniformity Inside Oven 0 1 C to 68 C Inside Bottles 0 1 C to 68 C Heating Element 500 Watts Shaker Tray Speed 54 106 cycles min Carousel Rotation 12 20 rom Lower Chambe
8. 69 01 Rev U Hybridization Ovens Introduction The HB 1000 Hybridizer is a cost effective high performance 10 bottle hybridization oven with state of the art microprocessor temperature and variable speed controls The HL 2000 HybriLinker System is designed with a combination of hybridizer and crosslinker components This equipment is intended to be used in the laboratory environment The HM 4000 Multidizer System is designed as a multifunction unit with a variety of motion options The lower chamber operates in the same manner as the HB 1000 However the HM 4000 has an additional upper chamber for the use of a reciprocating shaker tray an orbital rocker tray or an acrylic carousel Refer to the Accessories section of this manual for ordering information The HB 1000 Hybridizer can be set to operate anywhere from 10 to 15 RPM enabling consistent saturation of samples for washing or hybridizing Having the ability to remove the rotary carousel wheel and replace it with an interchangeable rocker plate enables all hybridization protocols in one reliable benchtop unit An easily removable protective tray under the carousel allows easy clean up of spilled media A touch sensitive keypad and microprocessor provide accurate temperature control from ambient 10 C to 99 9 C The keypad is located just below a large LED readout Once the desired hybridization temperature is entered the readout displays the current temperature inside the cha
9. C relieve the pressure by unscrewing and re tightening the cap If using Radioactive material see the Decontamination section of this manual CAUTION The HL 2000 HybriLinker System and HM 4000 Multidizer System are designed to be carried or lifted by two people Page 3 81 0169 01 Rev U Hybridization Ovens Hybridization Oven Specifications HB 1000 Hybridizer Hybridization Oven Part Numbers Volts Hz 95 0030 01 115V 60Hz 95 0030 02 230V 50Hz 95 0030 03 100V 50Hz Specifications Net Weight 43 lbs Temperature Ambient 10 C to 99 9 C Temperature Stability Inside Oven 0 3 C to 68 C Inside Bottles 0 1 C to 68 C Temperature Uniformity Inside Oven 0 1 C to 68 C Inside Bottles 0 1 C to 68 C Temperature Display LCD Heating Element 1250 watts Rotation Speed Off 10 to 15 RPM Bottle Capacity Twenty 20 35 x 150 mm Ten 10 35 x 300 mm Dimensions Exterior 17 5W x 16H x 16D in 445 x 406 x 406mm Interior 14W x 10 75H x 10 75D in 856 x 273 x 273mm HL 2000 HybriLinker System Page 4 The HybriLinker System combines a hybridization oven and a crosslinker in one space saving unit Each section has separate power supplies for independent operation Part Numbers Volts Hz 95 0031 01 115V 60Hz 95 0031 02 230V 50Hz 95 0031 03 100V 50Hz Specifications Unit Net Weight 60 Ibs Dimensions Exterior 17 5W x 24H x 15D in 445 x 610 x 381mm Hybridization Oven Temperature Ambient
10. Hybridizer HybriLinker and Multidizer Hybridization Ovens Operating Instructions HL 2000 HybriLinker HB 1000 Hybridizer HM 4000 Multidizer IMPORTANT To familiarize yourself with the correct operation procedures read these instructions before operating your UVP Hybridizer Multidizer or HybriLinker system SUVP An Analytik Jena Company UVP LLC Ultra Violet Products Ltd Unit 1 Trinity Hall Farm Estate 2066 W 11th Street Upland CA 91786 Tel 909 946 3197 800 452 6788 Fax 909 946 3597 Web Site www uvp com Nuffield Road Cambridge CB4 1TG UK Tel 44 0 1223 420022 Fax 44 0 1223 420561 81 0169 01 Rev U Hybridization Ovens Table of Contents Table Of Contents cccccccccseeecceececceeeesseeeeeceeesseeceseueeeseeessaueessaeeeseneeesseeess 2 METRO GING TO ecasecte at cact tee sect E cacasetaace cance anes 3 Hybridization OVEN Specifications cccecccsecceeececeececeeeeceeeeseceseueeseeeeseeeas 4 SOTO MSU UIST ONS vesasadeseauate E nemvoutnnasnader neaweneanerantemboctuae 6 Operating ASMUGHONS persen a A E neeesanianeme 8 Gal eran Cleanih gesen sadansideuraeateeayedsidedereatieacsasnteurect lt 13 FIC DIAGCIMOMII IANS errama nea aoeamiceadse dave eae E 14 FAC CESSOMIES sonon esau ap a eartenidenuewstaehnulolieued come 16 Maintenance Repair and Technical Assistance cccecceeeceeeseeeeeeeeeeees 17 my DNGIZalion 1 ECNNIGUCS imeona cess ro aea a eii 18 Page 2 81 01
11. age 10 connectors ensure that the proper configuration of the male connector or the plug has been properly connected to the power cord Turn the On Off switch to the On position NOTE WHEN TURNED ON THE CROSSLINKER DEFAULTS TO THE LAST USED UV EXPOSURE SETTING The last UV exposure setting will now be displayed on the LCD and the last function setting will be noted by glowing red spot s on the display panel Place your sample requiring exposure into the chamber The Crosslinker can be operated on the following settings Preset Ultraviolet Energy Exposure 1 Press PRESET on the tactile touchpad Then press ENERGY The red light at each position should now be lit and the preprogrammed UV exposure setting of 120 000 microjoules per cm is displayed in the LED as 1200 Note The LCD displays 1200 This is in hundreds and therefore must be multiplied by 100 to obtain an exposure 2 Press START After a slight delay to energize the UV tubes the LCD will begin to count down The unit automatically stops at the end of the exposure cycle and will beep five times Exposure is now complete NOTE Though the preset UV exposure setting is factory set at 120 000 microjoules per cm it is possible to change this if necessary as follows a Press and hold PRESET on the tactile touchpad until you hear an audible signal Then press ENERGY on the touchpad b Set your new preset UV exposure by pressing the number
12. an aborted exposure press START on the touchpad The exposure will continue from the point at which the exposure was aborted e To reset an aborted exposure press RESET The LCD and touchpad will return to the last used setting e The unit will not operate with the door open Opening the door during a cycle aborts the cycle Reclosing the door will reset the cycle to the last entered cycle used To restart press the START key Changing the UV Wavelength of the Crosslinker The Crosslinker is shipped with shortwave UV tubes However should user requirements and applications change it is possible to modify the ultraviolet wavelength and recalibrate the UV sensor and microprocessor to the new UV wavelength This is accomplished by purchasing FIVE tubes of the new UV wavelength and the proper UVP calibration sensor TUBE P N WAVELENGTH SENSOR P N DESCRIPTION 34 0006 01 365 nm Longwave 97 0016 02 UVX 36 Longwave 34 0042 01 302 nm Midrange 97 0016 04 UVX 31 Midrange 34 0007 01 254 nm Shortwave 97 0016 01 UVX 25 Shortwave UV Wavelength Calibration Procedure 1 2 9 After disconnecting the unit from the electrical supply install the five new tubes Plug the corresponding UV calibration sensor into the back of the Crosslinker chamber just below the pull out tray Place the UV calibration sensor in the middle of the tray of the exposure chamber Close the Crosslinker door Invoke the calibration mode of the Cross
13. arousel for 10 50ml Conical Bottles 76 0075 01 Carousel Acrylic for HM 4000 upper chamber 76 0089 01 Capacity 4 35 x 300mm 8 35 x 150mm or 8 50mm Conical Other Equipment Rocker Tray for all models 12 5W x 10D in 318 x 254 mm 98 0067 03 Removable plate is manufactured of stainless steel Orbital Motion Tray for all models 10 3 W x 8 2 D 98 0067 04 Specification 30 rom 2 Angle For use of the orbital tray outside the ovens order the following Orbital Motion Tray external stand alone 100 115V 98 0067 05 Orbital Motion Tray external stand alone 230V 98 0067 06 Specification 30 rom 2 Angle Rocker Tray Orbital Motion Tray Page 16 81 0169 01 Rev U Hybridization Ovens Maintenance Repair and Technical Assistance 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 UVP LLC In the US toll free 800 452 6788 or 909 946 3197 Fax 909 946 3597 2066 W 11th Street Upland CA 91786 USA Ultra Violet Products Ltd In Europe UK call 44 0 1223 420022 Fax 44 0 1223 420561 Ultra Violet Products Ltd Unit 1 Trinity Hall Farm Estate Nuffield Road Cambridge CB4 1TG UK NOTE A Returned Goods Authorization RGA number must be obtained from UVP Customer Service prior to returning any product to UVP Warranty UVP LLC warrants its produ
14. cts to be free of defects in materials and workmanship for a period of one 1 year from date of purchase The foregoing warranty of UVP shall be of no force and effect if buyer has modified or damaged the product Bulbs are warranted for 90 days All warranties or merchantability and fitness for any purpose and all other warranties expressed or implied except those expressly set forth herein are deemed waived and excluded UVP s duty under the warranty is limited to replacement and or repair of the defective part at the option of UVP LLC UVP shall not be held liable for any expenses or damages incurred by the purchaser except expressly set forth herein and in no event shall UVP be liable for any special incidental or consequential damages of any kind This warranty does not supersede any statutory rights that may be available in certain countries Page 17 81 0169 01 Rev U Hybridization Ovens Hybridization Techniques There are really two main steps to a hybridization reaction hybridizing two strands of complementary 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 in fact successful binding is proportional to their concentration The concentration of the target nucleic acid is the independent variable in all hybridizati
15. e 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 81 0169 01 Rev U Hybridization Ovens Protocol 1 Random Priming Method for Tagging DNA with Fluorescein Labeled 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 uL Nucleotide mix 300um each of dAT P dCTP dGTP and 60um dTTP Random nonamer 9 mer primers 2 5 ug uL in water Reaction buffer 10X 50mM MgCl2 10mM 2 Mercaptoethanol 500 mM Tris HCl pH 7 5 Tagged nucleotide fluorescene 11 dUTP Template DNA in water 5ng mL Procedure 1 OF ey Page 20 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 Place the tube into the boiling water bath for 5 minutes Immediately place tube on ice for 5 minutes Centrifuge for 15 seconds in microcentrifuge Add the reagents listed below to a fresh tube on ice in the follo
16. ilter in 0 15X SSC 0 1 SDS for 15 minutes at 50 C repeat once Store blots in 2X SSC at room temperature 81 0169 01 Rev U Hybridization Ovens Radioactive Probes Additionally you will need e X Ray film holder e X Ray film e 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 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 Inthe 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 Page 22 81 0169 01 Rev U Hybridization Ovens Protocol 3 Chemiluminescence Detection HRP Tagged Alkal
17. ine Phosphatase AP Probes or Antibody Conjugates Equipment e Clear plastic cling wrap or Clear transparent sheet protector e UVP EC3 or AC1 Darkroom with Cooled CCD camera e Pipette Reagents for Chemiluminescence e ECL or other detection reagent 1 e ECL or other if required detection reagent 2 e Membrane following hybridization Procedure 1 Mix equal volumes of detection reagents 1 and 2 2 Pipette the mixture over the surface of the membrane and leave at room temperature for 1 minute 3 Drain the sample and wrap it transfer to Darkroom 4 Close the darkroom and image using CCD camera References Ross J Nucleic Acid Hybridization Essential Techniques 1998 John Wiley and Sons ISBN 0 471 97125 1 Sambrook J Molecular cloning a Laboratory manual 1987 Cold Spring Harbor Laboratory Press ISBN 0 87969 309 6 Current Protocols in Molecular Biology 1987 John Wiley and Sons ISBN 0 471 50338 X HybriLinker and Multidizer are trademarks of UVP LLC Page 23 81 0169 01 Rev U
18. ization Ovens Replacement Parts Replacement parts lists are provided below for the HB 1000 Hybridizer and HL 2000 HybriLinker systems Repairs or replacement other than as specified in the following procedures should only be completed by authorized service personnel Replacement Parts for HB 1000 Hybridizer DESCRIPTION Borosilicate Glass Bottle with Cap and O Ring Large 35 x 300mm Cap and O Ring for all Bottle sizes Bottle Holder Carousel Carousel Drive Shaft Rod Drip Tray Fuses Main Switch Knob Motor Control Adjustable Rubber Foot Door Handle Compression Latch Power Cord 115V Power Cord 230V UK Power Cord 230V Euro QTY NEEDED N po I N h Replacement Parts for HL 2000 HybriLinker Crosslinker Page 14 DESCRIPTION Borosilicate Glass Bottle with Cap and O Ring Large 35 x 300mm Cap and O Ring for all Bottle sizes Bottle Holder Carousel Carousel Drive Shaft Rod Drip Tray Tubes 254nm Shortwave Ballasts 100V Ballasts 115V Ballasts 230V Starters 100V Starters 115V Fuses Main Switch Knob Motor Control Adjustable Rubber Foot Door Handle Compression Latch Micro Switch Door Interlock Power Cord 115V Power Cord 230V UK Power Cord 230V Euro QTY NEEDED N N a Aa CE o_o PART NO 07 0194 02 88 0004 01 76 0070 01 76 0064 03 10 0292 01 Contact UVP 53 0165 01 72 0030 01 72 0062 02 22 0110 01 58
19. linker by holding down STOP on the tactile touchpad and then turning the power to the unit ON A sequence of tones will be heard from the Crosslinker and a 180 second count will display on the LCD once the calibration procedure has begun No calibration occurs during this 180 second period This time sequence allows for the UV tube sources to warm up and stabilize Upon completion of the 180 second period measurements are automatically made with the UV calibration sensor and the Crosslinker sensor These measurements are compared to limits of acceptability If sensor readings are unacceptable an error code 01 02 03 or 04 will flash on the LCD Press any key to stop 10 Check all connections and perform system calibration once again If the same error message appears Page 11 81 0169 01 Rev U Hybridization Ovens call UVP 11 This calibration process should be repeated following any unsuccessful recalibration If calibration is unsuccessful replace the new UV wavelength tubes with the previously removed old wavelength tubes 12 Successful recalibration to the NEW UV wavelength changes the values in the microprocessor and the numeric setting value is displayed on the LCD To return to original or another wavelength the proper UV calibration sensor is needed Page 12 81 0169 01 Rev U Hybridization Ovens Care and Cleaning NOTE Always unplug the unit from the electrical supply before cleaning or drying the unit
20. lizes shortwave 254nm ultraviolet energy for many applications and protocols The crosslinker s internal microprocessor measures and controls UV output ensuring maximum energy efficiency UV crosslinking takes seconds as compared to hours for oven baking The UV crosslinking procedure optimizes the use of the ultraviolet energy while ensuring that crosslinking is consistent WARNING The crosslinker is a powerful source of ultraviolet UV radiation Even though it is not easily accessible do not attempt to disengage or override the internal safety interlocks Exposure to UV radiation may result If the UV sources remain on when the door is open the unit is malfunctioning and use should be discontinued until the unit has been serviced Do not expose unprotected eyes or skin to UV radiation NOTE This equipment is not intended for interconnection with any other devices Use of this equipment other than as intended may create a safety hazard and or malfunction 1 Place the unit on a level working surface Be sure there is adequate room in front of the door to open it easily 2 Plug the female end of the power cord into the unit 3 Plug the male end of the power cord into a properly grounded electrical outlet The proper operating voltage of the Crosslinker is found on the product information label Note For 230V models or those requiring special power cord Crosslinker Control Panel Page 9 81 0169 01 Rev U Hybridization Ovens P
21. mber The chamber environment is calibrated at 68 0 C At this temperature the accuracy is 0 5 C An internal circulating fan assists in uniformly distributing heating throughout the chamber Two different hybridizer bottle sizes are available for use within the hybridizer using the standard carousel provided with the unit The rotary carousel wheel can hold up to twenty 20 35 x 150 mm bottles or ten 10 35 x 300 mm bottles In addition combinations of both size bottles may be used at the same time An optional carousel kit for oversized bottles is available There may be build up of pressure within the hybridization bottles when they are taken from ambient to hybridization temperature To help in relieving some of this pressure preheat the solutions and boitles Also be sure bottles are opened at the same temperature at which the hybridization was done Do not allow the bottles to cool before opening To ensure the hybridization bottles remain leak proof and pressure proof prevent temperatures above 70 C If temperatures above this are used without first relieving the pressure within the bottle there is a risk that bottles will leak and or break due to internal pressure build up If the bottles are accidentally taken above 70 C without relieving the pressure DO NOT open the hybridizer door Turn off the hybridizer and allow it to cool before opening the door and checking the contents of the oven Should the bottles be used above 70
22. on reactions Since the target concentration is usually the unknown variable an excess of labeled probes 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 However with an enormous amount of probe around in the 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 is to wash it 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 Step 1 Nucleation slow whereby the two separate nucleic acid strands come into 1 jek i close proximity of each other A duplex region is formed y TE where a minimum of three bases of one strand complements y A to those on the second strand If the remainder of the strands p k E are complementary the two strands will anneal or zipper together very quickly The rate limiting step in nucleic acid Step 2 Zippering fast hybridization is the duplex formation which again explains why probe to target concentrations are critical mei g PN _ ae ae g Experimental Protocols There
23. ottle Positioning Page 6 81 0169 01 Rev U Hybridization Ovens Installing the Rollers and Optional Trays To insert rollers in the hybridization oven carousel slide the rollers into the receptacle on the left side of the chamber first followed by the right side To install the Reciprocating Shaker Tray standard with the HM 4000 place the tray over the rollers in the hybridization oven To install the Rocker Tray optional on all units remove the metal carousel from the chamber Place the tray into the chamber with the coupling arm to the right side Place the coupling sleeve over the motor drive shaft such that the pins on the inside of the coupling sleeve engage the slots in the motor drive shaft of the chamber Rotate the sleeve counter clockwise 30 until locked in place To remove the tray reverse the process To install the Orbital Motion Tray optional on all units open the chamber door remove the carousel connect the power cable to the orbital tray and to the power port inside the unit and place the tray in the oven so the switch is accessible Turn the hybridization oven Motor and Power switches ON and turn the orbital tray Power switch ON Rocker Tray Orbital Motion Tray Reciprocating Shaker Tray Page 7 81 0169 01 Rev U Hybridization Ovens Operating Instructions Please note that this equipment is not intended for interconnection with any other devices Use of this equipment other than intended may c
24. r Same as the HB 1000 Hybridizer 81 0169 01 Rev U Hybridization Ovens Setup Instructions Two different bottle sizes are available for use within the hybridizer The rotary wheel carousel can hold up to twenty 20 35 x 150 mm bottles or ten 10 35 x 300 mm bottles In addition combinations of both bottle sizes may be used at the same time e To insert bottle holder carousel Fig 1 slide the two wheels onto the rod and tighten the thumb screws Fig 2 Insert the rod into the holder on the right wall of the hybridizer chamber Then slide the rod into the holder on the left wall of the chamber e To adjust for various bottle lengths loosen the thumb screws on the bottle clip wheel and adjust to desired width e To insert bottles into bottle clips gently push the bottle into the clips Fig 3 Insert one side at a time for large bottles e f offset skewed bottle positioning is desired Fig 4 loosen the thumb screw for only one bottle clip wheel Slightly rotate the wheel until the required angle is obtained Retighten the thumb screw Note Offset or skewed bottle positioning can allow for better mixture of bottle contents NOTE ALWAYS USE AN EVEN NUMBER OF BOTTLES AND LOAD BOTTLES OPPOSITE EACH OTHER TO BALANCE THE BOTTLE HOLDER THIS PREVENTS EXCESSIVE WEAR OF THE BEARINGS AND DRIVE MOTOR Fig 1 Inserting Bottle Holder Carousel Fig 2 Adjusting for Bottle Lengths Fig 3 Inserting Bottles Fig 4 Offset Skewed B
25. reate a safety hazard and or malfunction Using the Hybridization Oven 1 Place the unit on a level working surface and provide adequate room in front of the door to open it easily Plug the female end of the power cord into the unit To turn on the main power supply depress the Power switch toward ON on the right side of the control panel To adjust the temperature setting to the necessary degree press either the UP or DOWN buttons below the temperature control display to raise or lower the displayed degree displayed in degrees Celsius This setting is adjustable from ambient 10 C to 99 9 C See Setting the Temperature Controls for more detail To turn on the motor for the bottle holder press the motor control power switch to the ON position To adjust the bottle carousel to the desired RPM simply turn the motor control knob clockwise to increase the RPM and counterclockwise to decrease the RPM RPM Motor Temp Main Control ON OFF Setting ON OFF Minimum RPM OFF 10 Maximum RPM 18 NOTE The inside of the hybridizer should be cleaned with a damp cloth The bottle carousel assembly can be cleaned with soap and water 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
26. s on the touchpad Remember The number indicated on the LCD display must be multiplied by 100 to determine the proper exposure setting The new setting will then appear on the display c Press ENTER on the touchpad The new setting is now installed Preset Ultraviolet Time Exposure 1 Press PRESET and then TIME on the tactile touchpad The red light at each position should now be lit and the preprogrammed UV exposure time of 2 minutes should be displayed on the LCD 2 Press START on the touchpad After a slight delay to energize the UV tubes the LCD will begin to count down NOTE THE TIME EXPOSURE IS SET IN MINUTES AND TENTHS OF A MINUTE 3 The unit will automatically stop at the end of the exposure cycle and will beep five times Exposure is now complete NOTE Though the PRESET ULTRAVIOLET TIME EXPOSURE is factory set to 2 minutes it is possible to change this if necessary as follows a Press and hold PRESET on the touchpad until you hear an audible signal Then press TIME b Set your new preset UV time exposure by pressing the numbers on the tactile touchpad The new setting will appear on the LED display c Press ENTER and the new setting will be installed User Set UV Energy Exposure Sometimes it may be necessary for you to set your own standards for exposure This is easily accomplished as follows 1 Press ENERGY on the tactile touchpad then set your energy exposure requiremen
27. ts by pressing the numbers corresponding to your desired UV exposure time on the touchpad The energy exposure settings should now be displayed as flashing on the LCD NOTE Your energy exposure settings displayed must be multiplied by 100 If the settings are correct press ENTER on the touchpad 2 Press START on the touchpad After a slight delay to energize the UV tubes the LCD will begin to count down The unit will stop automatically at the end of the exposure cycle and will beep five times Exposure is now complete 81 0169 01 Rev U Hybridization Ovens User Set UV Time Exposure Setting your own UV time exposure can be achieved as follows 1 Press TIME on the tactile touchpad and then set your requirements by pressing the corresponding numbers Your time exposure settings will be displayed flashing on the LCD Remember that your time exposure settings are displayed in minutes and tenths of a minute If your settings are correct press ENTER on the tactile touchpad 2 Press START After a slight delay to energize the UV tubes the LCD will begin to count down The unit will stop automatically at the end of the exposure cycle and will beep five times Exposure is now complete 3 Atthe end of the exposure cycle open the door and remove the sample Operational Notes e To abort an exposure press STOP on the tactile touchpad The LCD will display the remaining exposure time e To restart
28. uce 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 Situation Response G C 45 55 Follow normal protocol G C lt 45 Lower salt and temperature G C gt 55 Raise salt and temperature Evidence of probe Lower salt and temperature target mismatching Target and probe is degraded Hybridize in a formamide based buffer on aqueous solution Unacceptable high background Use less probe 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 M the temperature at which the half of the duplex molecules will dissociate 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 TM is reduced by 0 65 C Therefore at 80 formamide reactions can b
29. wing order 10 mL Nucleotide mix 5 mL Tagged nucleotide 5 mL Reaction buffer x10 5 mL Random primers 10 mL Boiled DNA 14 mL Water 1 mL DNA polymerase Mix gently and incubate at 37 C for 1 hour i Stop the reaction by adding 2 mL EDTA j Store probes at 20 C in the dark 7O 7 Oo 2900 p 81 0169 01 Rev U Hybridization Ovens 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 40 50 for 2 hours or until dissolved Store at 20 SDS will precipitate at room temperature but remain in solution at 37 UVP Hybridization bottle s and caps 15 mL plastic tube Boiling Water Bath Bucket of ice
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