マニュアル Hybridization oven/shaker
Contents
1. 3 0 1 Remove all packaging and place the Hybridization Oven Shaker on a level working surface ensuring that there is sufficient room above the instrument to allow the door to be opened fully 3 0 2 Plug the female end of the power cable into the Hybridization Oven Shaker 3 0 3 Connect the power cable to a suitably grounded electrical outlet The correct operating voltage of the Hybridization Oven Shaker is found on the product information label on the rear of the instrument 3 0 4 Turn the Mains ON OFF switch 1 on Fig 1 to the ON position 3 0 5 The GE Healthcare Hybridization Oven Shaker is now ready for use 1 Mains ON OFF switch 6 Oven OVER TEMP dial 2 Temperature ON OFF switch 7 Rotisserie shaker ON OFF switch 3 Temperature SET switch 8 Rotisserie shaker speed selector dial 4 Temperature SET dial 9 Oven FAULT LED 5 Digital temperature display 3 1 Fig 1 Diagram of instrument control panel 3 2 Setting the oven temperature 3 2 1 Press the Mains ON OFF switch 1 see Fig 1 to the ON position 3 2 2 Press the Temperature ON OFF switch 2 to the ON position 3 2 3 Press the Temperature SET switch 3 at the same time rotate the Temperature SET dial 4 until the required temperature is shown on the digital display 5 3 2 4 Release the Temperature SET switch 3 3 2 5 Rotate the Oven OVER TEMP dial 6 to the SET temperature 5 oC NOTE The automatic temperature cut out operates at 12. oC above the SET temperature to ensure that the desired temperature is maintained at all times The OVER TEMP setting acts as an instrument fail safe mechanism Should the OVER TEMP setting be reached the red FAULT LED 9 will light up denoting a fault in the temperature control system 3 2 6 The oven will now automatically heat up the the SET temperature 6 5 1 2 3 4 7 8 9 6 3 3 Setting up the rotisserie The rotisserie is installed in the Hybridization Oven Shaker during transit To use the rotisserie for bottle hybridization the following procedure should be adopted 3 3 1 Lift up the oven door to its fullest extent to allow complete access to the oven interior 3 3 2 Lift the rotisserie vertically out of the oven and place on the bench 3 3 3 Place the membranes to be hybridized in to the required number of hybridization bottles Using the rotisserie as a stand for the bottles place the bottles into the rotisserie pushing them down as far as they will go NOTE Always ensure that the weight is evenly distributed on both sides of the rotisserie Place an empty hybridization bottle into the other side of the rotisserie as a counterbalance if necessary 3 3 4 Place the rotisserie into the voen onto the rotation mechanism ensuring that the serrated bands at either end of the rotisserie locate onto the steel cogs of the rotation mechanism at the rear of the oven The plastic flanges of the rotisserie locate
3. 110 3000 AA0004 PB10204 a 1 2 amp 3 30 800 AA0084 PB10384 a 1 2 amp 3 15 400 AA0064 PB10164 a 1 2 amp 3 32P dCTP 10 mCi ml 220 6000 AA0075 PB10475 a 1 2 amp 3 110 3000 AA0005 PB10205 a 1 2 amp 3 30 800 AA0085 PB10385 a 1 2 amp 3 15 400 AA0065 PB10165 a 1 2 amp 3 32P dGTP 10 mCi ml 110 3000 AA0006 PB10206 a 1 2 amp 3 30 800 AA0086 PB10386 a 1 2 amp 3 15 400 AA0066 PB10166 a 1 2 amp 3 32P dTTP 10 mCi ml 110 3000 AA0007 PB10207 a 1 2 amp 3 30 800 AA0087 PB10387 a 1 2 amp 3 15 400 AA0067 PB10167 a 1 2 amp 3 32P ATP 10 mCi ml 110 3000 PB10200 a 1 2 amp 3 15 400 PB10160 a 1 2 amp 3 32P CTP 10 mCi ml 110 3000 PB10202 a 1 2 amp 3 20 mCi ml 30 800 PB20382 b 1 2 amp 3 15 400 PB10162 a 1 2 amp 3 40 mCi ml 30 800 PB40382 e 3 32P GTP 10 mCi ml 110 3000 PB10201 a 1 2 amp 3 15 400 PB10161 a 1 2 amp 3 32P UTP 10 mCi ml 110 3000 AA0003 PB10203 a 1 2 amp 3 20 mCi ml 30 800 PB20383 b 1 2 amp 3 15 400 PB10163 a 1 2 amp 3 40 mCi ml 30 800 PB40383 e 3 32P ATP 10 mCi ml gt 185 gt 5000 AA0018 PB10218 a 1 2 amp 3 2 mCi ml gt 185 gt 5000 PB218 c 1 2 amp 3 110 3000 AA0068 PB10168 a 1 2 amp 3 110 3000 PB
4. ensure that the spill tray is repositioned correctly and screwed down securely On starting the shaker the drive shaft will automatically re engage itself This may take a few revolutions of the drive mechanism Fig 3 Adjustment of angle of tilt of platform shaker 8 10 o tint 4 o tint central spindle 4 0 Hybridization using the platform shaker The Hybridization Oven Shaker is compatible with the hybridization technologies available from GE Healthcare These include radioactive hybridizations using Rapid hyb buffer and the range of non radioactive systems for the labelling and detection of proteins and nucleic acids see appendix 1 When using the platform shaker the hybridization and washing conditions recommended in the appropriate GE Healthcare literature should be used The following protocol therefore provides a guideline For specific hybridization times and temperatures refer to the relevant protocol booklet 4 0 1 Prepare blots as recommended in the appropriate Hybond protocol booklet 4 0 2 Set the oven temperature and over temperature values as described in section 4 2 4 0 3 Place the membrane in a suitable box or bag and cover with sufficient prehybridization buffer to ensure that the entire surface of the membrane is covered Recommended volume surface area ratio is given in most protocol booklets Seal the box or bag securely 4 0 4 Place the box or bag on the platform set the oscillation speed t
5. low activity waste awaiting disposal RPN1532 1 safe 400 x 240 x 220 mm 10 mm acrylic Beta midi waste safe designed to hold standard sharps RPN2038 1 safe 315 x 225 x 235 mm Beta mini waste safe compatible with the Beta work tank RPN2039 1 safe 150 x 120 x 100 mm Beta tip safe mini waste safe with hinged lid for safe pipette tip disposal RPN2081 1 safe 150 x 150 x 150 mm Beta tip safe plastic bags disposable draw string plastic bags RPN2083 pack of 25 Beta pipette guard designed for use with Gilson Pipetman TM range of pipettes for use with the P20 100 size RPN1544 1 guard for use with the P200 size RPN1545 1 guard for use with the P1000 size RPN1546 1 guard set of beta pipette guards RPN1556 3 guards RPN1544 RPN1545 RPN1546 Radiation safety accessories Safety tray a defined working area for bench radioactive work 685 x 455 mm RPN1534 1 tray 685 x 455 mm white RPN1533 1 tray 685 x 535 mm RPN2042 1 tray 530 x 330 mm RPN2043 1 tray 1120 x 535 mm RPN2063 1 tray 565 x 535 mm RPN2083 1 tray 455 x 255mm RPN2093 1 tray all other safety trays are yellow Appendix 7 Hybridization products 25 Product code Pack description Kodak TM APET safety tray liners similar chemical resistance to above liners but with none of the disposal problems associated with plastics such as PVC to match RPN1533 34 RPN1528 pack of 25 to match RPN2042 RPN2048 pack of 25
6. ml gt 37 gt 1000 SJ1303 1 amp 3 20 mCi ml gt 37 gt 1000 SJ603 i 1 amp 3 10 mCi ml 15 400 SJ263 1 amp 3 40 mCi ml 30 800 SJ40383 f 3 32S ATP S 10 mCi ml gt 37 gt 1000 SJ1318 1 amp 3 10 mCi ml 22 600 SJ318 1 NOTE Redivue 32P and 33P nucleotides contain dye and stabilizer in formulation A All 35S nucleotides are supplied in stabilized aqueous solution containing 20 mM DTT at 370 MBq ml 10 mCi ml except where stated Formulation decoder A Stabilized aqueous solution containing 5 mM 2 mercaptoethanol at 370 MBq ml 10 mCi ml B Stabilized aqueous solution containing 5 mM 2 mercaptoethanol at 740 MBq ml SP6 T7 grade C Ethanol water 1 1 now supplied in the CDC container at 74 MBq ml 2 mCi ml D Aqueous solution at 74 MBq ml 2 mCi ml E Stabilized aqueous solution at 1 5 GBq ml 40 mCi ml SP6 T7 grade F SP6 T7 Grade supplied at 1 5 GBq ml 40 mCi ml G Stabilized aqueous solution pH6 0 H Contains no DTT in situ grade I Stabilized aqueous solution containing 20 mM DTT at 740 MBq ml 20 mCi ml SP6 T7 grade Pack size key 1 9 25 MBq 250 Ci 2 18 5 MBq 500 Ci 3 37 MBq 1 mCi 22 Appendix 5 Non radioactive labelling and detection systems 23 Hyperfilm high performance autoradiography films Size Pack size Hyperfilm MP Hyperfilm ECL multipurpose film for use with autoradiography film enhanced chemiluminescence 18 x 24 cm 25 sheets RPN6K
7. platform and close the oven door 3 4 4 Ensure the Mains ON OFF switch 1 see figure 1 is in the ON position and that the desired temperature has been set see section 4 2 3 4 5 Turn the Rotisserie Shaker ON OFF switch 7 to the ON position 3 4 6 Rotate the Rotisserie Shaker speed selector dial 8 clockwise until the desired shaker speed is reached Allowable values are 5 70 strokes per minute The shaker platform will now oscillate at the set rate 7 3 4 7 When hybridization is complete turn the ON OFF switch 7 to the OFF position NOTE The shaker platform can be operated at a choice of tilt angles 4 o or 10 o The larger the angle the greater the vertical movement of the platform at the end of each stroke and hence the greater the agitation of the contents of the box This facility allows further fine tuning of the rocking motion of the shaker platform in addition to controlling the shaker speed The tilt angle is controlled by the position of the pegs in the rocker mechanism which is attached to the rear of the spill tray To alter the tilt angle unscrew the spill tray by rotating the nylon retaining screws anti clockwise Lift out the spill tray Remove the two pegs E in figure 2 and screw in to the appropriate position in the rocker mechanism Figure 3 below shows the relative positions of each hole and the tilt angle to which they correspond Fig 2 Hybridization oven drive components On completion
8. primary wash buffer solution at 55 C 3 x 5 min with secondary wash buffer at room temperature NOTE The room temperature washes can be achieved by switching off the oven and leaving the door open whilst performing the washes or by allowing the oven to cool down to room temperature before performing the final washes 5 3 6 Remove the membrane from the bottle and detect using the standard procedures outlined in the protocol booklet Gene Images Random Prime Hybridizations 5 3 7 Drain off the hybridization buffer rinse the bottle and the membrane thoroughly with 2x SSC and discard 5 3 8 Perform the following stringency washes in large volumes 100 ml minimum of the following solutions at a rotisserie speed of 8 rpm 2 x 10 min with 1x SSC 0 1 SDS at 60 oC 1 x 10 min with O 1x SSC 0 1 SDS at 60 oC 5 3 9 Remove the membrane from the bottle and detect using the standard procedures outlined in the booklet The detection procedure may be carried out using the shaker mode of the Hybridization Oven Shaker NOTE The room temperature washes can be achieved by switching off the oven and leaving the door open whilst performing the incubations or by allowing the oven to cool down to room temperature 12 6 0 Maintenance care cleaning of the oven shaker The GE Healthcare Hybridization Oven Shaker is designed to provide trouble free operation The base of the oven and the shaker tray act as a spills tray and will contain any
9. to match RPN2043 RPN2058 pack of 25 to match RPN2063 RPN2068 pack of 25 to match RPN2083 RPN2088 pack of 25 to match RPN2093 RPN2098 pack of 25 Work box insert compatible with work boxes and safe storage boxes Work box insert 1 5 ml tubes RPN1540 1 insert Work box insert 2 0 ml tubes RPN2035 1 insert Work box insert 0 5 ml tubes RPN2036 1 insert Microcentrifuge tube rack S shaped rack with lifting points to allow easy removal from boxes Microcentrifuge tube rack 1 5 ml tubes RPN1542 1 rack Microcentrifuge tube rack 0 5 ml tubes RPN2037 1 rack Redivial station designed to hold 2 Redivials and 16 microcentrifuge tubes RPN1585 1 workstation CDC storage box lockable storage box for RPN2032 1 box 4 GE Healthcare CDS s Radioactive spills kit wall mountable kit containing materials to clear up RPN2030 1 kit small radioactive spills Radioactive spills refill pack refill pack for Radioactive spills RPN2030 RPN2031 1 pack http www gehealthcare co jp lifesciences Alkphos Direct Amprep ECF ECL ECL Plus Gene Images Hybond Hypercassette Hyperfilm Hyperscreen Hypertorch KiloBase Megaprime Nick Rapid hyb Ready To Go Rediprime Redivial Redivue Sensitize and Trackertape are trademarks of GE Healthcare Bio Sciences Ltd or its subsidiaries GE Healthcare is a trademark of General Electric Company Kodak and APET polyethylene terephthalate are trademarks of Eastman Kod
10. 168 c 1 2 amp 3 10 mCi ml 1 11 30 PB10132 a 1 2 amp 3 2 mCi ml 0 11 3 0 PB108 c 1 2 amp 3 2 mCi ml 0 11 3 0 PB170 d 1 2 amp 3 32P GTP 10 mCi ml gt 185 gt 5000 PB10244 a 1 amp 3 32P ddATP 10 mCi ml gt 185 gt 5000 PB10235 a 1 amp 3 110 3000 PB10233 a 1 amp 3 32P ATP 2 mCi ml 1 11 30 PB171 d 1 2 amp 3 32P pCp 10 mCi ml 110 3000 PB10208 a 1 2 amp 3 32P NAD 10 mCi ml 37 1000 PB10282 g 1 amp 3 33P 32P ATP 10 mCi ml 37 110 1000 3000 AH9968 BF1000 a 1 2 amp 3 32P dATP 10 mCi ml 37 110 1000 3000 AH9904 BF1001 a 1 2 amp 3 32P UTP 20 mCi ml 37 110 1000 3000 BF1002 a 1 35S 35S dATP S 10 mCi ml gt 37 gt 1000 AG1000 SJ1304 1 amp 3 22 600 AG1001 SJ304 1 amp 3 15 400 AG1002 SJ264 1 amp 3 35S dCTP S 10 mCi ml gt 37 gt 1000 SJ1305 1 amp 3 22 gt 600 SJ305 15 400 SJ265 35S dATP S 10 mCi ml gt 37 gt 1000 SJ1334 h 1 amp 3 Appendix 4 Radioactive nucleotides 21 Compound Concentration Specific activity Redivue Standard Pack size TBq mmol Ci mmol product code product code see key 35S ATP S gt 37 gt 1000 SJ1300 22 600 SJ300 15 400 SJ260 35S CTP S 10 mCi ml gt 37 gt 1000 SJ1302 1 amp 3 40 mCi ml 30 800 SJ40382 f 3 35S UTP S 10 mCi
11. Hybridization oven shaker RPN2511 user manual hybridization Underwriters Laboratories Inc Listed E195497 3 Hybridization Oven Shaker RPN2511 Contents 1 0 Introduction 2 0 Specifications 3 0 Setting up the Hybridization Oven Shaker 4 0 Hybridization using the platform shaker 5 0 Hybridization using the rotisserie 6 0 Maintenance care cleaning of the instrument 7 0 Wiring diagram 8 0 Troubleshooting guide Appendices Associated GE Healthcare products 1 Products for electrophoresis 2 Hybond membranes for nucleic acid and protein applications 3 Systems for radioactive labelling of nucleic acids 4 Radioactive nucleotides 5 Systems for non radioactive labelling and detection 6 Products for autoradiography and chemiluminescent detection 7 Hybridization products 8 Radiation safety products IMPORTANT Please read the separate instructions for use before operating the GE Healthcare Hybridization Oven Shaker to familiarize yourself with its electrical installation and safety precautions 1 0 Introduction One of the most widely used techniques in the molecular biology field is the immobilization of DNA and RNA onto a solid support membrane and subsequent hybridizations with a specific single stranded probe labelled to facilitate its detection Using the GE Healthcare Hybridization Oven Shaker ensures that the temperature and shaking rotation frequency and hence the stringen
12. RPN2103K 18 x 43 cm 25 sheets RPN36K 30 x 40 cm 25 sheets RPN7K RPN2104K 35 x 43 cm 25 sheets RPN8K 35 x 43 cm 75 sheets RPN30K 20 cm x 25 m 1 roll RPN34K 18 x 24 inches 75 sheets RPN1675K RPN3103K 5 x 7 inches 25 sheets RPN1676K RPN1674K 8 x 10 inches 25 sheets RPN1677K RPN2114K 8 x 10 inches 75 sheets RPN1678K RPN3114K 10 x 12 inches 25 sheets RPN1681K Hypercassette TM cassettes for autoradiography and light detection Size Code standard Code deep 18 x 24 cm RPN11642 RPN1628 24 x 30 cm RPN11643 30 x 40 cm RPN11644 RPN1627 35 x 43 cm RPN11645 18 x 43 cm RPN11646 20 x 40 cm RPN11647 5 x 7 inches RPN11648 8 x 10 inches RPN11649 RPN1629 10 x 12 inches RPN11650 Hyperscreen TM intensifying screens for 32P and 1251 autoradiography Size Code Quantity 18 x 24 cm RPN1662 1 pair 24 x 30 cm RPN1663 1 pair 30 x 40 cm RPN1664 1 pair 35 x 43 cm RPN1665 1 pair 18 x 43 cm RPN1666 1 pair 20 x 40 cm RPN1667 1 pair 5 x 7 inches RPN1668 1 pair 8 x 10 inches RPN1669 1 pair 10 x 12 inches RPN1670 1 pair Miscellaneous Product name Code Hypertorch TM RPN1620 Battery powered LED darkroom torch pack of 3 Sensitize TM RPN2051 Optimized preflash system TrackerTape TM RPN2050 An adhesive waterproof tape that phosphoresces to give a permanent written image on film For use with radioactive or chemiluminescent emissions Appendix 6 Pro
13. ak Inc Saranwrap is a trademark of Dow Chemical Company Gilson Pipetman is a trademark of Gilson SA Underwriters Laboratories Inc Listed E195497 All goods and services are sold subject to the terms and conditions of sale of the company within the General Electric Company group which supplies them A copy of these terms and conditions is available on request GE Healthcare UK Ltd GE Healthcare Place Little Chalfont Buckinghamshire England HP7 9NA GE Healthcare Bio Sciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden GE Healthcare Bio Sciences Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA GE Healthcare Europe GmbH Munzinger Strasse 9 D 79111 Freiburg Germany um S1449 00 06 Rev C 2000 06
14. cy of hybridization and washing steps are rigidly controlled This enables rapid and reproducible probing of nucleic acids and proteins immobilized on nylon and nitrocellulose membranes The GE Healthcare Hybridization Oven Shaker is a multipurpose instrument combining accurate temperature control with a choice of interchangeable hybridization modes Variable speed rotisserie holding 7 x 35 mm or 2 x 70 and 2 x 35 mm hybridization bottles A variable speed platform shaker for box hybridizations depurination denaturation and neutralization steps The instrument is Economical bottle hybridization minimizes probe volumes reducing reagent volumes and enhancing signal intensity Precise stringency of hybridization and washing steps are rigidly controlled ensuring reproducible results Sensitive validated protocols ensure optimal hybridization and washing steps enhancing multiple reprobing when using Hybond TM membranes Safe the double glazed polycarbonate acrylic door offers excellent thermal insulation whilst minimizing radiation exposure Convenient small foot print maximizes the use of limited laboratory space The instrument is suitable for use in conjunction with radiolabelled probes using Rapid hyb TM buffer non radioactive nucleic acid labelling and detection systems such as AlkPhos DirectTM and protein labelling and detection systems including ECLTM and ECL Plus TM Some protocols for use with these applicati
15. ducts for autoradiography and chemiluminscent detection 24 Product name Pack size Product code Rapid Hyb buffer Rate enhanced hybridization buffer 125 ml RPN1635 for use with radiolabelled nucleic acid probes 500 m RPN1636 Hybridization buffer tablets 100 tablets RPN131 Each tablet makes 10 ml of hybridization buffer for use with any nucleic acid probe Appendix 8 Radiation safety products For the safe handling and storage of 32P Product code Pack description Beta shielding is manufactured from 10 mm heavy gauge optical quality acrylic which effectively absorbs 3 2P beta particles Beta starter pack comprehensive shielding and containment RPN2052 1 pack package for 32P users Beta safety screen 15 o ideal when working seated at the bench RPN1536 1 screen Beta safety screen 45 o when working standing up RPN1537 1 screen Beta side rear screen use vertically or horizontally to protect your colleagues RPN2034 1 screen Beta workbox for the storage of low level radioactive working RPN1539 1 box Beta workbox insert for the storage of 32 microcentrifuge tubes RPN1540 1 insert Beta safe storage box holds 3 tube racks or workstations RPN1541 1 box Beta work tank beta workstation gives 360 o protection from beta radiation RPN2033 1 worktank Beta heavy duty tube rack 35 mm acrylic with 4 tube holes RPN1543 1 rack Beta waste safe safe short term storage of
16. ied out in the bottles If the platform shaker is used follow the standard washing procedure mentioned in the appropriate protocol booklet If bottles are to be used it is necessary to modify the standard washing procedure Outlined in this section are the optimized washing protocols for radioactive hybridizations and non radioactive based hybridizations Radioactive Hybridizations 5 3 1 Carefully drain off the hybridization buffer rinse the bottle and the membrane thoroughly with 2x SSC and discard 5 3 2 Perform the following stringency washes in large volumes 100 ml minimum of the following solutions at a rotisserie speed of 8 rpm 2 x 10 min with 2x SSC 0 1 SDS at 65 C 1 x 15 min with 1x SSC 0 1 SDS at 65 C 2 x 10 min with 0 1x SSC 0 1 SDS at 65 C NOTE This last step is a high stringency wash and should be omitted if related sequences are to be probed More washes over the same time period for each stringency condition can improve background 5 3 3 Remove the membrane from the bottle drain off excess stringency wash wrap in SaranWrap TM and autoradiograph 11 rotation of membrane AlkPhos Direct Hybridizations 5 3 4 Drain off the hybridization buffer rinse the bottle and the membrane thoroughly with primary wash buffer and discard 5 3 5 Perform the following stringency washes in large volumes 100 ml minimum of the following solutions at a rotisserie speed of 8 rpm 3 x 10 min with
17. nd tight plus a quarter turn DO NOT OVERTIGHTEN or the thread of the cap can be damaged leading to leakages NOTE If placing several small blots into one bottle prewet the membranes as above and space them out along the length of the bottle with forceps 5 1 4 Place the bottle on a flat surface and roll it gently in the opposite direction to that which the membrane is coiled This rolling action causes the membrane to uncoil so lining the inner surface of the bottle NOTE The use of a mesh in bottle hybridizations to ensure uniform contact between the membrane surface and the buffer has been recommended However studies at GE Healthcare laboratories using a wide range of hybridization mesh technologies demonstrate a resulting loss of sensitivity due to partial absorption of the probe into the mesh A nylon mesh RPN2519 is available as an optional extra as it can facilitate easier handling of a number of blots and the more fragile nitrocellulose membranes These handling advantages should be considered against the potential loss of sensitivity before use When a hybridization mesh is used in conjunction with the membrane the following procedure should be adopted 5 1 5 Pre wet the mesh alongside the membrane in 2x SSC buffer and place the prewetted membrane on top of the mesh The mesh should be slightly larger than the blot in all dimensions Roll both up together with the mesh on the outside of the membrane and insert int
18. o 30 strokes min and prehybridize for the required length of time 4 0 5 Remove the box or bag from the oven and carefully add the labelled single stranded probe denaturation may be required post labelling refer to the appropriate protocol booklet to the prehybridization buffer NOTE Do not pipette the probe solution directly on to the membrane as this may cause localized background 4 0 6 Reseal the box or bag securely replace it on the platform and hybridize for the required length of time 4 0 7 Remove the membranes and place in a clean box containing the first stringency wash solution 4 0 8 Increase the oscillation speed of the platform to 60 strokes min Carry out the recommended washing protocol at the appropriate temperature and for the appropriate times 9 5 0 Hybridization using the rotisserie Several advantages are associated with performing hybridizations in bottles namely those of safety and economy as outlined in the introduction However the use of bottles for hybridizations and washing procedures requires certain adaptations to standard protocols 5 1 Assembly of membranes into bottles 5 1 1 Add approximately 20 ml 2x SSC buffer into the hybridization bottle The rotisserie acts as a convenient bottle stand 5 1 2 Pre wet the membrane in 2x SSC buffer and loosely roll it up 5 1 3 Insert the rolled up membrane into the bottle and replace the cap Ensure that the cap is screwed on securely ha
19. o the bottle as described above 6 1 4 5 2 Hybridization 5 2 1 Set the required oven temperature and over temperature as detailed in section 4 2 5 2 2 Discard the 2x SSC used in the bottle to unroll the membrane and replace with prehybridization buffer Recommended volumes are provided in most GE Healthcare protocol booklets Generally a minimum of 10 15 ml per 20 x 20 cm blot is advised Seal the bottle avoiding overtightening 5 2 3 Place the hybridization bottle s into the rotisserie as detailed in section 4 3 add counterbalance bottles if necessary Place the rotisserie into the oven so that the bottles are rotating in the same direction as the membrane is rolled see Figure 4 overleaf 10 Fig 4 Rotation of rotisserie 5 2 4 Prehybridize the membrane for the specified length of time at a rotisserie speed of 4 rpm 5 2 5 Following prehybridization turn off the rotisserie remove the rotisserie from the oven Add the labelled probe to the buffer either by removing an aliquot of the buffer adding the probe and returning the aliquot to the bottle or by adding the probe directly into the bottle avoiding the membrane 5 2 6 Hybridize for the specified length of time at a rotisserie speed of 4 rpm 5 3 Membrane washing procedures Membranes can either be removed from the hybridization bottles and washed in a box on the platform shaker this is a more efficient procedure or the washing procedure may be carr
20. on to the small wheels on the oven floor Close the oven door 3 3 5 Ensure that the Mains ON OFF switch 1 is in the ON position and that the desired temperature has been set see section 4 2 3 3 6 Turn the Rotisserie Shaker ON OFF switch 7 to the ON position 3 3 7 Turn the Rotisserie Shaker speed selector dial 8 clockwise until the desired rotation speed is reached allowable values are 2 10 rpm The rotisserie will now start to rotate at the set rate 3 3 8 When hybridization is complete turn the Rotisserie Shaker ON OFF switch 7 to the OFF position 3 4 Setting up the platform shaker During transit the platform is stored vertically at the rear of the oven chamber It can remain in this position whilst the rotisserie is in use To use the platform shaker for sandwich box hybridizations the following procedure should be adopted 3 4 1 Open the oven door to its fuIlest extent Iift the rotisserie vertically and store in a safe place 3 4 2 Lift the platform by its handle A see figure 2 from its storage position slide it forward and locate it on the rocking mechanism by placing the side pegs 8 of the platform into the retainers C on the side walls of the oven chamber This action seats the nylon blocks D on the underside of the platform on to the pegs E protruding from the rocker mechanism at the rear of the oven 3 4 3 Place the box in which the hybridization is being performed on to the shaker
21. ons are included in section 6 4 2 0 Specification Overall dimensions Height 9 5 24 0 cm Depth 10 0 25 0 cm Width 11 25 28 5 cm Oven dimensions Height 8 0 20 0 cm Depth 9 0 23 0 cm Width 10 0 25 0 cm Weight 24 kg Capacity nominal 18 litres Temperature range Ambient plus 5 oC 80 oC Temperature precision 0 5 oC Temperature fluctuation 0 1 C 37 C Power rating 250 W Over temperature cut out 1 oC over set temperature Temperature variation lt 0 25 oC Rotisserie speed 2 10 rev min Shaker platform speed 5 70 strokes min Total angle of tilt 4 o or 10 o angle Electrical Nominal Voltage Hertz Amp Product code 230 V 50 Hz 3 1 A RPN2510 110 V 120 V 60 Hz 4 A RPN2511 100 V 50 60 Hz RPN2512 Each instrument is supplied with 1 rotisserie RPN2514 6 hybridization bottles RPN2516 and an instruction manual Accessories Rotisserie holds 7 x 35 mm hybridization bottles RPN2514 Rotisserie RPN2515 holds 2 x 70 mm and 2 x 35 mm hybridization bottles Hybridization bottle 230 x 35 mm RPN2516 For rotisserie RPN2514 pack of 6 Hybridization bottle 150 x 35 mm RPN2517 For rotisserie RPN2514 pack of 6 Hybridization bottle 230 x 70 mm RPN2518 For rotisserie RPN2515 pack of 2 Hybridization mesh 1 roll RPN2519 21 5 cm x 10 m 5 3 0 Setting up the Hybridization Oven Shaker
22. spillage that occurs during hybridization and washing procedures To ensure lasting operation the following instructions should be followed ALWAYS DISCONNECT THE HYBRIDIZATION OVEN SHAKER FROM THE ELECTRICAL SUPPLY BEFORE CLEANING OR DRYING THE INSTRUMENT 1 Any leakage from the hybridization bottles or the sandwich boxes should be cleaned up immediately Do not allow any liquids to enter the drive mechanism 2 Wipe away any liquids from inside and outside of the unit using soap and water with a soft cloth or sponge 3 Do not allow chemicals to remain on unit surfaces 4 Never clean unit with abrasive pads or cleaners 5 Never clean unit with acetone or chloroform 13 14 7 0 Wiring diagram 15 8 0 Troubleshooting guide This section briefly summarizes some of the potential problems encountered during membrane hybridizations More complete troubleshooting guides are supplied in the pack leaflet that accompany each product Symptom Cause Remedy Membrane curling up in hybridization Incorrect orientation of Ensure membrane is rolled up in the bottle bottle in rotisserie same direction as the bottle is rotating see section 6 2 High background Probe concentration too high Reduce probe concentration Unincorporated 32P nucleotides Remove unincorporated nucleotides not removed eg using Amprep TM C18 column Insufficient blocking Use recommended hybridization buffer or extend prehybridiza
23. tion time Insufficient washing Increase number of buffer changes during the washing stage or increase stringency of final wash Weak signal No transfer from gel to Load extra lanes with control DNA membrane Transfer can be checked by restaining gel with ethidium bromide If large DNA fragments are detected poorly use a depurination step 0 25 M HCI Probe not labelled Check probe labelling before hybridization See protocol in probe labelling booklet Probe not denatured Boil probe for 5 minutes before adding to hybridization buffer Low specific activity of probe Review labelling conditions Washes too stringent Increase buffer salt concentration and decrease temperature Patchy backgrounds Hybridization buffer or wash Increase volume of hybridization wash solution not evenly covering or solutions or use mesh see section membrane 6 1 4 Damaged membrane Handle membrane carefully with forceps High background around edge of Damaged membrane Use clean scissors or a sharp scalpel membrane to cut membrane 16 17 Appendix 1 Products for electrophoresis 18 Appendix 2 Hybond membranes for nucleic acid applications 19 Appendix 3 Radioactive labelling systems 20 Compound Concentration Specific activity Redivue product Standard product Pack size TBq mmol Ci mmol code code see key 32P 32P dATP 10 mCi ml 220 6000 AA0074 PB10474 a 1 2 amp 3
Download Pdf Manuals
Related Search