GE RPN2510E User's Manual
Contents
1. Use in a well ventilated area Ambient temperature range 5 C to 40 C Altitude to 2000 m Relative humidity not exceeding 80 Mains supply fluctuation not exceeding 10 Over voltage category II IEC60364 4 443 Pollution degree 2 Use with a minimum distance all around of 200 mm from walls or other items The unit should be carried using both hands Never move or carry the unit when in use or connected to the mains electricity supply In the case of mains interruption a fault or electrical failure the unit will continue to operate on restoration of the electricity supply or removal of the fault 3 1 Electrical Installation WARNING Lethal voltages inside the Hybridization Oven casing Always THIS INSTRUMENT MUST BE switch the Hybridization Oven Shaker off and remove the plug from the D EARTHED electrical socket before performing any cleaning or maintenance of the instrument IMPORTANT The Hybridization Oven Shaker has been supplied with a cordset and plug Check that the appropriate plug for your electrical supply has been included If there is any doubt consult a qualified electrician and inform your local GE Healthcare sales office Should the plug require replacement in the future the following information should be noted The wires in the mains lead are coloured in accordance with the following code GREEN YELLOW Earth BLUE Neutral BROWN Live As the colours of the wires in the mains lead of this item may not corr2. at a rotisserie speed of 8 rpm 3 x 10 minutes with primary wash buffer solution at 55 C 3 x 5 minutes 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 procedures outlined in the protocol booklet RPN2510EUM Chapter 7 Rev C 06 2007 T1 8 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 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 Only spare parts supplied or specified by GE Healthcare or its agent should be used Fi
3. Chapter 5 Rev C 06 2007 7 Fig 1 Diagram of instrument control panel Rotisserie shaker controls 1 Rotisserie shaker ON OFF button 2 Rotisserie shaker speed selector dial 3 Motor on indicator Temperature controls 4 Temperature on off button 5 Press to set button 6 LED temperature display 7 Temperature selector dial Note the Oven is fitted with automatic digital over temperature protection see Section 8 1 for more details 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 Place the rotisserie into the oven 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 B on figure 2 The plastic flanges of the rotisserie locate on to the small wheels on the oven floor Close the oven door Ensure that the desired temperature has been set see section 5 1 The speed controls are on the left hand side of the control panel numbers 1 to 3 in figure 1 Turn the Rotisserie Shaker ON by pressing the on off button 1 The red indicator light 3 above the speed selector dial 2 will illuminate Turn the Rotisserie Shaker speed selector dial 2 clockwise until the desired rotation speed is reached allowable values are 2 10 rpm The rotisseri
4. Waste Safes 400 x 240 x 220 mm 1 safe RPN1532 315 x 229 x 230 mm 1 safe RPN2038 150 x 120 x 100 mm 1 safe RPN2039 150 x 150 x 150 mm 1 safe RPN2082 Waste Safe Bags to fit RPN1532 25 bags RPN2091 to fit RPN2038 25 bags RPN2090 to fit RPN2082 25 bags RPN2089 Beta Work Tank 540 x 400 x 500 mm 1 tank RPN2033 Redivial workstation 160 x 90 x 75 mm 1 workstation RPN1585 Work Boxes and Safe Storage Boxes Work Boxes Beta Work Box 185 x 115 x 80 mm 1 box RPN1539 Beta Safe Storage Box 300 x 185 x 155mm 1 box RPN1541 Work Box Inserts 32 x 1 5 ml tubes 1 insert RPN1540 32 x 2 0 ml tubes 1 insert RPN2035 32 x0 5 ml tubes 1 insert RPN2036 RPN2510EUM Appendix 7 Rev C 06 2007 20 21 GE Healthcare GE Healthcare offices GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden GE Healthcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Healthcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Healthcare Bio Sciences KK Sanken Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan For contact information for your local office please visit www gelifesciences com contact GE Healthcare Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK http www gehealthcare com lifesciences imagination at work RPN2510EUM Rev C 06 2007
5. 0 120 V 60 Hz 4 A 100 V 50 60 Hz 4 A 5 70 strokes minute Product code RPN2510E RPN2511E RPN2512E Each instrument is supplied with 1 rotisserie RPN2514 6 hybridization bottles RPN2516 and an instruction manual Accessories Rotisserie holds 7 x 40 mm hybridization bottles Rotisserie holds 2 x 75 mm and 2 x 40 mm hybridization bottles Hybridization bottle 260 x 40 mm For rotisserie RPN2514 pack of 6 Hybridization bottle 170 x 40 mm For rotisserie RPN2514 pack of 6 Hybridization bottle 240 x 75 mm For rotisserie RPN2515 pack of 2 Hybridization mesh 1 roll 21 5 cm x 10 m RPN2510EUM Chapter 4 Rev C 06 2007 RPN2514 RPN2515 RPN2516 RPN2517 RPN2518 RPN2519 5 Setting up the Hybridization Oven Shaker 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 2 Leave the unit to stand for a minimum of 3 hours This will allow acclimation to the new ambient temperature 3 Plug the female end of the power cable into the Hybridization Oven Shaker 4 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 5 Turn the Mains ON OFF switch 1 on Fig 1 to the ON position 6 The GE Healthcare Hybridization Oven Shaker is no
6. 18 x 24cm 50 28 9068 43 Hyperfilm MP 18 x 24cm 100 28 9068 44 Hyperfilm MP 8 x 10 inches 50 28 9068 45 Hyperfilm MP 8 x 10 inches 100 28 9068 46 Hyperfilm MP 24 x 30 cm 50 28 9068 47 Hyperfilm MP 35 x 43 cm 50 28 9068 48 Hyperfilm MP Enveloped 18 x 24cm 50 28 9068 50 Hypercassette cassettes for autoradiography and light detection Size Code standard Code deep 18 x 24cm RPN11642 RPN11628 24x 30cm RPN11643 30 x 40 cm RPN11644 RPN11627 35x 43cm RPN11645 18 x 43 cm RPN11646 20 x 40cm RPN11647 5 x 7 inches RPN11648 8 x 10 inches RPN11649 RPN11629 10 x 12 inches RPN11650 Hyperscreen intensifying screens for 32P and 12 autoradiography Size Code Quantity 18 x 24 cm RPN1662 1 pair 24 x 30cm RPN1663 1 pair 30 x 40 cm RPN1664 1 pair 35x 43cm 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 Related Products Product name Description Code Hypertorch Battery powered LED darkroom torch pack of 3 RPN1620 Sensitize Optimized preflash system RPN2051 RPN2510EUM Appendix 5 Rev C 06 2007 18 Appendix 6 Hybridization buffer Product name Pack size Code Rapid Hyb buffer Rate enhanced hybridization buffer 125 ml RPN1635 for use with radiolabelled nucleic acid probes 500 m RPN1636 RPN2510EUM Appendix 6 Rev C 06 2007 19 Appendix 7 Radiation safety products Product Description Product
7. Dimensions Quantity Code Number CDC storage box 240 x 140 x 130 mm 1 box RPN2032 Pipette guards Beta pipette guard Gilson P20 100 1 guard RPN1544 Beta pipette guard Gilson P200 1 guard RPN1545 Beta pipette guard Gilson P1000 1 guard RPN1546 Multipack 1 each of the above codes 3 guards RPN1556 Beta Radiation safety starter packs 1 pack RPN2052 Radioactive spills kit Radioactive spills kit 1 kit RPN2030 Radioactive spills kit US only 1 kit RPN2030US Refill pack for radioactive spills kit 1 pack RPN2031 Refill pack for radioactive spills kit US only 1 pack RPN2031US Safety screens Beta safety screen 15 530 x 355 mm 1 screen RPN1536 Beta safety screen 45 600 x 355 mm 1 screen RPN1537 Beta side rear screen 550 x 350 mm 1 screen RPN2034 Safety trays and liners Safety Trays 685 x 455 mm 1 tray RPN1534 685 x 530 mm 1 tray RPN2042 530 x 330 mm 1 tray RPN2043 1120 x 535 mm 1 tray RPN2063 565 x 535 mm 1 tray RPN2083 455 x 255 mm 1 tray RPN2093 Safety Tray Liners to match tray 685 x 455 mm 25 liners RPN1528 to match tray 685 x 530 mm 25 liners RPN2048 to match tray 530 x 330 mm 25 liners RPN2058 to match tray 1120 x 535 mm 25 liners RPN2068 to match tray 565 x 535 mm 25 liners RPN2088 to match tray 455 x 255 mm 25 liners RPN2098 Beta heavy duty tube rack 96 x 35 x 50mm 1 rack RPN1543 Microcentrifuge tube racks 160 x 90 x 75 mm for 1 5 ml tubes 1 rack RPN1542 160 x 90 x 75 mm for 0 5 ml tubes 1 rack RPN2037 Beta Waste Safes and Bags Beta
8. GE Healthcare Amersham Hybridization oven shaker Product User Manual Codes RPN2510E RPN2511E RPN2512E Page finder 9 Legal Introduction Safety warnings and precautions 3 1 Electrical Installation Specification Setting up the Hybridization Oven Shaker 5 1 Setting the oven temperature 5 2 Setting up the rotisserie 5 3 Setting up the platform shaker Hybridization using the platform shaker Hybridization using the rotisserie 7 1 Assembly of membranes into bottles 7 2 Hybridization 7 3 Membrane washing procedures 7 3 1 Radioactive Hybridizations 7 3 2 AlkPhos Direct Hybridizations Maintenance care cleaning of the oven shaker 8 1 Error codes faults Troubleshooting guide Appendix 1 Products for electrophoresis Appendix 2 Hybond membranes for nucleic acid applications Appendix 3 Radioactive labeling systems Appendix 4 Non radioactive labeling and detection systems Appendix 5 Products for autoradiography and chemiluminscent detection Appendix 6 Hybridization buffer Appendix 7 Radiation safety products RPN2510EUM Pagefinder Rev C 06 2007 2 O ONNN DOD UH fF W PRPrPrPPR PrPrRPOOO Pe Nm Mh PP e Pp BP N OD WO fF WW 18 19 20 1 Legal GE imagination at work and GE monogram are trademarks of General Electric Company Amersham Alkphos Direct ECF ECL ECL Plus ECL Advance Hybond Hypercassette Hyperfilm Hyperscreen Hypertorch Megaprime N
9. carried 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 7 3 1 Radioactive Hybridizations 1 Carefully drain off the hybridization buffer rinse the bottle and the membrane thoroughly with 2 x SSC and discard Perform the following stringency washes in large volumes 100 ml minimum of NOTE This last step is a high stringency the following solutions at a rotisserie speed of 8 rpm wash and should be omitted if related 2 x 10 minutes with 2 x SSC 0 1 SDS at 65 C sequences are to be probed 1x 15 minutes with 1 x SSC 0 1 SDS at 65 C 2 x 10 minutes with 0 1 x SSC 0 1 SDS at 65 C More washes over the same time period for each stringency condition can improve background 3 Remove the membrane from the bottle drain off excess stringency wash wrap in SaranWrap and autoradiograph 7 3 2 AlkPhos Direct Hybridizations 4 Remove the membrane from the bottle and detect using the standard Drain off the hybridization buffer rinse the bottle and the membrane thoroughly with primary wash buffer and discard Perform the following stringency washes in large volumes 100 ml minimum of the following solutions
10. dom prime dCTP only 25ng 10 minutes 2x 10 membrane RPN1633 hybridization RPN1634 Ready To Go Random prime dCTP only 10 ng 1 ug 5 minutes 2 x 10 membrane 27 9240 01 DNA labelling hybridization beads DNA labelling Megaprime Random prime any dNTP 25 ng 10 minutes 2 x 10 membrane RPN1604 hybridization RPN1605 RPN1606 RPN1607 Nick translation Nick translation any dNTP Lug 2 3 hours 2 x 10 production of N5000 large amounts N5500 RPN2510EUM Appendix 3 Rev C 06 2007 16 of probe Appendix 4 Non radioactive labeling and detection systems Labelling and detection Sensitivity Time from Duration of Strip before Quantification Recommended application system hybridization light output re probing to detection AlkPhos Direct RPN3690 0 06 pg 1 hour 5 days yes no Single copy Southern and Northerns ECL Direct RPN3000 0 5 pg 1 hour 1 2 hours no no High target applications eg colony plaques RPN2510EUM Appendix 4 Rev C 06 2007 17 Appendix 5 Products for autoradiography and chemiluminscent detection Hyperfilm high performance autoradiography films Product description size number sheets code Hyperfilm ECL 5 x 7 inches 50 28 9068 35 Hyperfilm ECL 18 x 24cm 50 28 9068 36 Hyperfilm ECL 18 x 24cm 100 28 9068 37 Hyperfilm ECL 8 x 10 inches 50 28 9068 38 Hyperfilm ECL 8 x 10 inches 100 28 9068 39 Hyperfilm ECL 24 x 30 cm 50 28 9068 40 Hyperfilm ECL 35 x 43 cm 50 28 9068 41 Hyperfilm MP 5 x 7 inches 50 28 9068 42 Hyperfilm MP
11. e supplied in the pack leaflet that accompany each product Symptom 1 Membrane curling up in hybridization bottle 2 High background 3 Weak signal 4 Patchy backgrounds 5 High background around edge of membrane RPN2510EUM Chapter 9 Rev C 06 2007 Cause 1 1 2 1 2 2 2 3 2 4 3 1 3 2 3 3 3 4 3 5 4 1 4 2 5 1 Incorrect orientation of bottle in rotisserie Probe concentration too high Unincorporated 32P nucleotides not removed Insufficient blocking Insufficient washing No transfer from gel to membrane Probe not labeled Probe not denatured Low specific activity of probe Washes too stringent Hybridization buffer or wash solution not evenly covering membrane Damaged membrane Damaged membrane 13 Remedy 1 1 2 1 2 2 2 3 2 4 3 1 3 2 3 3 3 4 3 5 4 e 4 2 5 1 Ensure membrane is rolled up in the same direction as the bottle is rotating see section 7 2 Reduce probe concentration Remove unincorporated nucleotides Use recommended hybridization buffer or extend prehybridization time Increase number of buffer changes during the washing stage or increase stringency of final wash Load extra lanes with control DNA 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 Check probe labelling before hybridi
12. e will now start to rotate at the set speed 8 When hybridization is complete turn the Rotisserie Shaker OFF by pressing the on off button 1 The red indicator 3 will go out 5 3 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 1 Open the oven door to its fullest extent lift the rotisserie vertically and store in a safe place 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 of the platform into the retainers D on the side walls of the oven chamber This action seats the nylon blocks C on the underside of the platform on to the pegs E protruding from the rocker mechanism at the rear of the oven Place the box in which the hybridization is being performed on to the shaker platform and close the oven door Ensure that the desired temperature has been set see section 4 1 Turn the Rotisserie Shaker ON by pressing the on off button 1 The red indicator light 3 above the speed selector dial 2 will illuminate Rotate the Rotisserie Shaker speed selector dial 2 clockwise until the desired shaker speed is reached Allowable values are 5 70 strokes per min
13. espond with the coloured markings identifying the terminals in your plug proceed as follows e The wire which is coloured green yellow must be connected to the terminal in the plug which is marked with the letter E or by the earth symbol or colours green or green yellow e The wire which is coloured blue must be connected to the terminal which is marked with the letter N or coloured black e The wire which is coloured brown must be connected to the terminal which is marked L or coloured red e Fit the plug with the appropriate fuse Always replace the plug cover never use the plug without the cover The GE Healthcare Hybridization Oven Shaker is intended for research use only and not for any other purpose IF IN DOUBT CONSULT A QUALIFIED ELECTRICIAN RPN2510EUM Chapter 3 Rev C 06 2007 5 4 Specification Overall dimensions Height 17 43 5 cm Depth 15 38 0 cm Width 15 38 0 cm Oven dimensions Height 8 20 0 cm Depth 9 23 0 cm Width 10 25 0 cm Weight 24 kg Capacity nominal 18 litres Temperature range Ambient plus 5 C 80 C Temperature precision 0 5 C Temperature fluctuation 0 1 C 37 C Power rating 250 W Over temperature cut out 1 C over set temperature Temperature variation lt 0 25 C Rotisserie speed 2 10 rev minute Shaker platform speed Electrical Nominal Voltage Hertz Amp 230 V 50 Hz 3 1A 11
14. ick Rapid hyb Ready To Go Rediprime Sensitive and Trackertape are trademarks of GE Healthcare companies All third party trademarks are the property of their respective owners 2003 2007 General Electric Company All rights reserved First published November 2003 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information http www gehealthcare com lifesciences GE Healthcare UK Limited Amersham Place Little Chalfont Buckinghamshire HP7 9NA UK RPN2510EUM Chapter 1 Rev C 06 2007 3 2 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 labeled to facilitate its detection Using the GE Healthcare Hybridization Oven Shaker ensures that the temperature and shaking rotation frequency and hence the stringency 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 hybridizatio
15. klets Generally a minimum of 10 15 ml per 20 x 20 cm blot is advised Seal the bottle avoiding overtightening Place the hybridization bottle s into the rotisserie as detailed in section 5 2 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 3 rotation of membrane RPN2510EUM Chapter 7 Rev C 06 2007 10 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 NOTE The use of a mesh in bottle hybridizations to ensure uniform contact between the membrane surface and the buffer has been recommended Fig 3 Rotation of rotisserie Prehybridize the membrane for the specified length of time at a rotisserie speed of 4 rpm Following prehybridization turn off the rotisserie remove the rotisserie from the oven Add the labeled 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 Hybridize for the specified length of time at a rotisserie speed of 4 rpm 7 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
16. n modes e Variable speed rotisserie holding 7 x 40 mm or 2 x 75 and 2 x 40 mm hybridization bottles e A variable speed platform shaker for box hybridizations depurination denaturation and neutralization steps The instrument is e Economical bottle hybridization minimizes probe volumes reducing reagent volumes and enhancing signal intensity e Precise stringency of hybridization and washing steps are rigidly controlled ensuring reproducible results e Sensitive validated protocols ensure optimal hybridization and washing steps enhancing multiple reprobing when using Hybond membranes e Safe the double glazed polycarbonate acrylic door offers excellent thermal insulation whilst minimizing radiation exposure e Convenient small foot print maximizes the use of limited laboratory space The instrument is suitable for use in conjunction with radiolabeled probes using Rapid hyb buffer non radioactive nucleic acid labeling and detection systems such as AlkPhos Direct and protein labeling and detection systems including ECL ECL Plus and ECL Advance Some protocols for use with these applications are included in section 7 RPN2510EUM Chapter 2 Rev C 06 2007 4 3 Safety warnings and precautions If the equipment is not used in the manner described in this manual the protection provided might be impaired This equipment is designed to operate under the following conditions For indoor use only
17. nd NX Hybond ECL Hybond C Extra Hybond P 82mm 50 discs RPN82B RPN82S RPN82N RPN82T RPN82D RPN82E 87mm 50 discs RPN87B RPN87S RPN87N RPN87T 132 mm 50 discs RPN132B RPN132S RPN132N RPN132T RPN132D 137 mm 50 discs RPN137B RPN137S RPN137N RPN137T RPN137E 82 mm 50 gridded discs RPN1782B 87 mm 50 gridded discs RPN1787B 132 mm 50 gridded discs RPN1732B 137 mm 50 gridded discs RPN1737B 30 x 50 cm 5 sheets RPN3050B RPN3050S RPN3050N 9 x 10 cm 10 sheets RPN910D 10 x 10 cm 10 sheets RPN1010D 15 x 20 cm 10 sheets RPN1520B RPN1520S RPN1520 RPN1520T RPN1520D 20 x 20 cm 10 sheets RPN2020B RPN2020S RPN2020 RPN2002T RPN2020D RPN2020E RPN2020F 22 2 x 22 2 cm 10 sheets RPN2222B RPN2222S RPN2222 14x 16 cm 15 sheets RPN1416F 12 x 10 cm 20 sheets RPN1210B RPN1210S RPN1210 15 x 10 cm 20 sheets RPN1510B RPN1510S RPN1510 6x8cm 50 sheets RPN68D 7x8cm 50 sheets RPN78D 11 9 x 7 8 cm 50 sheets RPN119B RPN119S RPN119N 22 2 x 22 2 cm 50 sheets RPN2250B 22 5 x 22 5 cm 50 sheets RPN225B 115 x 73 mm 50 sheets RPN1576B 20cmx3m 1 roll RPN203B RPN203S RPN203N RPN203D RPN203E 30cm x 3m 1 roll RPN303B RPN303S RPN3O3N RPN3O3T RPN303D RPN3O3E RPN3O3F 30cmx3m 1 roll RPN3032D a 0 2 um pack size RPN2510EUM Appendix 2 Rev C 06 2007 15 Appendix 3 Radioactive labeling systems Labelling Technology Nucleotide Amount of Labelling Probe specific Recommended Product system template time activity application code dpm ug Rediprime II Ran
18. quarter turn DO NOT OVERTIGHTEN or the thread of the cap can be damaged leading to leakages Place the bottle on a flat surface and roll it gently in the opposite direction to that in which the membrane is coiled This rolling action causes the membrane to uncoil so lining the inner surface of the bottle 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 Pre wet the mesh alongside the membrane in 2 x 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 into the bottle as described above 7 1 4 i 2 Hybridization 1 Set the required oven temperature as detailed in section 5 1 Discard the 2 x SSC used in the bottle to unroll the membrane and replace with prehybridization buffer Recommended volumes are provided in most GE Healthcare protocol boo
19. rehybridize for the required length of time 5 Remove the box or bag from the oven and carefully add the labeled single NOTE Do not pipette the probe solution stranded probe denaturation may be required post labelling refer to the directly on to the membrane as this appropriate protocol booklet to the prehybridization buffer may cause localized background 6 Reseal the box or bag securely replace it on the platform and hybridize for the required length of time 7 Remove the membranes and place in a clean box containing the first stringency wash solution 8 Increase the oscillation speed of the platform to 60 strokes minute Carry out the recommended washing protocol at the appropriate temperature and for the appropriate times RPN2510EUM Chapter 6 Rev C 06 2007 9 7 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 7 1 Assembly of membranes into bottles 1 Add approximately 20 ml 2 x SSC buffer into the hybridization bottle The rotisserie acts as a convenient bottle stand Pre wet the membrane in 2 x SSC buffer and loosely roll it up Insert the rolled up membrane into the bottle and replace the cap Ensure that the cap is screwed on securely hand tight plus a
20. tting of non approved parts may affect the performance of the safety features designed into the instrument 8 1 Error codes faults The Oven has built in fault diagnostics If a fault occurs this system will display an error code in the LED display to help the service engineer rectify the problem Please see the table below for details of error codes and other faults In the unlikely event that a problem does occur note down which code fault you observe and contact your nearest GE Healthcare Office for further assistance If you have your own service personnel available for repair a comprehensive service manual is available on request Fault Temperature probe PT100 reading low Safety system in operation Automatic over temp system activated Fault displayed by LED displays Err and 0 1 alternatively Temperature probe PT100 reading high Automatic over temp system activated LED displays Err and 0 2 alternatively Triac fault Rotisserie shaker motor stalled Secondary relay control activated Cuts power to motor Top left dot flashes in display No movement seen but Rotisserie red indicator light still on Software crash RPN2510EUM Chapter 8 Rev C 06 2007 Cuts mains power No temperature or motor motor power all LED s blank 9 Troubleshooting guide This section briefly summarizes some of the potential problems encountered during membrane hybridizations More complete troubleshooting guides ar
21. ute The shaker platform will now oscillate at the set speed 7 When hybridization is complete turn the Rotisserie Shaker OFF by pressing the on off button 1 The red indicator will go out 3 Fig 2 Hybridization oven drive components RPN2510EUM Chapter 5 Rev C 06 2007 8 6 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 1 Prepare blots as recommended in the appropriate Hybond protocol booklet 2 Set the oven temperature as described in section 5 1 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 Place the box or bag on the platform set the oscillation speed to 30 strokes minute as described in section 5 3 P
22. w ready for use 1 2 3 4 5 6 Hybridisation Oven Shaker 5 1 Setting the oven temperature 1 The Oven temperature controls are located on the right hand side of the control panel numbers 4 to 7 in figure 1 2 To turn the temperature control ON press the on off button 4 The LED display 6 will show the current Oven temperature 3 To set the temperature press and hold the press to set button 5 and at the same time rotate the temperature selector dial 7 until the required temperature is shown on the digital display 6 4 Release the press to set button 5 and the temperature display 6 will revert back to the actual Oven temperature 5 The set temperature can be viewed at any time simply by pressing and holding the press to set button 6 6 The oven will now automatically heat up to the set temperature 5 2 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 1 Lift up the oven door to its fullest extent to allow complete access to the oven interior 2 Lift the rotisserie vertically out of the oven Unpack and place on the bench 3 Place the membranes to be hybridized into 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 RPN2510EUM
23. zation See protocol in probe labelling booklet Boil probe for 5 minutes before adding to hybridization buffer Review labelling conditions Increase buffer salt concentration and decrease temperature Increase volume of hybridization wash or solutions or use mesh see section 7 1 step 4 Handle membrane carefully with forceps Use clean scissors or a sharp scalpel to cut membrane Appendix 1 Products for electrophoresis DNA Markers Precise Sizing Digest of Natural DNAs ladder ladder DNA marker 27 4060 01 27 4005 01 27 4007 01 27 4004 01 5009 20009 10000 231305 450 1900 8000 9416 400 1800 6000 6557 350 1700 5000 4361 300 1600 4000 2322 250 1500 3000 2027 200 1400 2500 1353 150 1300 2000 1078 100 1200 1500 872 50 1100 1000 603 1000 500 5644 900 310 800 281 700 271 600 234 500 194 400 125 300 118 200 72 100 a not necessarily the largest size fragment possible only the largest that is readily distinguishable b cos ends are located on these bands c may not always be visible Marker code number Recommended gel Loading amount yg lane Heat before loading 27 4005 01 2 agarose 6 polyacrylamide 2 0 No 27 4007 01 1 5 agarose 2 0 No 27 4004 01 0 8 agarose 0 5 No 27 4060 01 1 agarose 0 5 1 0 60 65 C for 2 minutes RPN2510EUM Appendix 1 Rev C 06 2007 14 Appendix 2 Hybond membranes for nucleic acid applications Size Pack size Hybond N Hybond XL Hybond N Hybo
Download Pdf Manuals
Related Search