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User Manual - Laboratory ovens

1. 1 CHAPTER 2 NUCLEIC ACID TRANSFER PROCEDURES 3 eer e e esses aaa saa a OS 3 Colony Plaque Blotting AEN 4 tne amo 5 5 Capillary Blotting EE 6 Figure 2 1 Capillary Blot Assembiy 7 Northierm BloHIrig tote oca toot ct dte oct tret nts 8 CHAPTER 3 HYBRIDISATION PROCEDURES censere 9 Southern Blot DNA Hvbdtsatons sss 9 Northern Blot RNA Hybridisation sss 11 Notes for Nucleic Acid Hybridisations using the Thermo Range of elc p a aa aaa 12 Placing Membranes in a Bopien 12 Figure 3 Inserting Membranes in a Bottle 14 PrehyDridIsalOE een tinae dote add edid debutant 15 FAV ORIGIS QUO Mes e aa R ace 15 le E 16 CHAPTER 4 NUCLEIC ACID PROBES 18 Double Stranded DNA Probes 18 Single Stranded DNA Probes 18 RNA Probes I 19 Synthetic Oligonucleotides sss 19 CHAPTER 5 NON RADIOACTIVE 5 81 20 The DIG System in Thermo Hybridisation Ovens 20 Electrophoresis A Transier n 21 DNA Hybridisation a dates 21 CHAPTER 6 HYBRIDISATION USING OLIGONUCLEOTIDE PROBES 23 Thermo Scie
2. PHP SCREW AB POINT 1 23 130048 Md INTERNAL TOOTH LOCK WASHER 14 24 130091 M4 SS FLAT WASHER H Thermo Scientific Exploded Drawing Model 6240 6241 6242 6243 6244 6245 Shake N Stack Oven 6240 200 1 B Rev 1 Page 2 of 2 Shake n Stack Section 8 Technical Specifications SHEET 10F2 137023 FOR M DE 137024 FOR MODE Exploded Dravving Model 6240 6241 6242 6243 6244 6245 Shake N Stack Oven Page 1 of 2 6240 200 2 B Rev 1 8 6 Shake n Stack Thermo Scientific Section 8 Technical Specifications 2 03091 01301 ONIYdS S ZN 660061 166 2 1 11 2 2 030910 43813 aay 901 60081 16 22 NJHSVM 1911 SS nl 160061 o 2 0309 0 X201 HLOOL TYNE31N SS N 470061 62 x sw 660001 82 2 ONIY 9NINIVI3U 9013201 41391 110099 12 2 104 X3H Gen 690019 92 LNN ml Y80019 Io 30328 did H 170066 ve 1 308 did SS yn one 62 2 3825 4 4 wwe 241 660066 22 118Y2 1031002 31 3 1 62006 le 118Y2 1 Nol 9 62006 02 118Y2 830004 SNIVM Seoose 6 ANNOY X WN9 SS 39084 0001014 102062 8 9407 4021 ONTGNL 30021118 vunn L 18001118 33N11 N3AO 1 16 620622 9 1904419 N3A0 910222
3. Solution Volume Final Concentration 20 x SSPE 5 x SSPE 100 x Denhardt s 0 5ml 5 x Denhardt s reagent 10 SDS 0 5 SDS Add denatured salmon sperm DNA to 100ug ml NB All solutions should be prepared in clean sterile glassware using distilled water and highest quality reagents NB In addition Hybridisation membranes should always be handled wearing gloves or using forceps Where it is necessary to sterilise the membranes place between sheets of filter paper and autoclave for 15 minutes Thermo Scientific May 2003 Issue 7 31 APPENDIX Il FACTORS AFFECTING STRINGENCY OF HYBRIDISATION REACTIONS The stringency of Hybridisation determines the degree to which mismatched hybrids are permitted to form The optimum Hybridisation temperature for DNA DNA Hybridisations is 25 C below the melting temperature Tm The Tm is affected by 1 The concentration of monovalent cation M 2 Base composition percentage G C content 3 The length of the duplex in base pairs L 4 The amount of formamide in the reaction mixture F These variables have been linked by the following equation see Meinkoth and Wahl 1984 Tm 81 5 C 16 6 logM 0 41 0 61 96 F 500 L This equation relates to probes longer than approximately 50 nucleotides see Chapter 6 for conditions relating to oligonucleotide Hybridisations As an illustration in a reaction carried out in a solution of 6 x SSC and no fo
4. P N 222057 Nylon Mesh Five large mesh sheets 9 x 9 in 23 x 23cm EE DIN 222053 Five small mesh sheets 4 x 6 in 10 x 15cm VES Ee P N 222052 Thermo Scientific Shake n Stack 8 3 Section 8 Technical Specifications Cee i Exploded Drawing Model 6240 6241 6242 6243 6244 6245 Shake N Stack Oven Q N 6240 200 1 B Rev 1 Page 1 of 2 8 4 Shake n Stack Thermo Scientific Section 8 Technical Specifications BILL OF MATERIALS d PART NO PART DESCRIPTION QTY 16041 8 INSULATED RING TERMINAL 2 02017 DOOR GASKET FOR SHAKE N STACK 3 3021 BLACK ADJUSTABLE FOOT M8X25 4 4 3022 NON SLIP SHOE 1 5 25024 SPACER 4 6 40382 FRONT LABEL 1 40620 INSULATION BACK 8 40621 INSULATION LEFT SIDE BOTTOM 9 40622 INSULATION RIGHT SIDE 0 40623 INSULATION TOP 40625 INSULATION LEFT SIDE TOP 2 1222013 16 1 JOVEN COVER SUB ASSEMBLY 3 1222013 31 4 JOVEN COVER BACK 4 231225 CONTROLLER GASKET 5 231229 DIGITAL TEMPERATURE CONTROLLER 6 310186 PANEL MOUNT POTENTIOMETER 7 360258 ROCKER SWITCH WITH GREEN INDICATOR 8 360259 ROCKER SWITCH WITH BLACK INDICATOR 9 360260 SPLASH PROOF SWITCH COVER 2 20 590043 M4 X 10mm SS PHP SCREW 7 2 610084 M4 SS HEX NUT 5 22 680021 M3 X MIO
5. 31 13402 831130 1 76 6 10222 7 8113881 83111 N3A0 1 L1 610222 Wuvi 110222 2 LAVHS 010222 171102 3140 600222 10 10011018 11 08 900222 6 191388 024 1031402 NIYA 01116 9 WOLLOG oi enen 717 1 Ove 831131 UVINENL 2016 9 2 0001 Wu 339Y4 ANNOY WHE 8012 6 y ONOT 2 339Y4 99 88 L O nl 29012 y 7 180441 09406 1112812 94142011 62062 c 1384039 GI Wr Nc 9 1607 2 2 4112 9018451 sen 110 8 9 ON Lud on S Y d31YN 40 1114 zo z eBeg 8 2 002 0729 U AQ 4915 N 9xeus Greg preg vco 29 729 Orc9 JepolN 60 0 pepojdx3 8 7 Shake n Stack Thermo Scientific Section 8 Technical Specifications E 4 2 156130 FOR MODELS 6240 6242 56131 FOR MODELS 6241 6243 6244 6245 Co Mod 230206 FOR MODELS 6240 6242 230210 FOR MODELS 6241 6243 6244 6245 5 FOR 220V MODELS NA 6240 6242 ONLY Exploded Drawing Model 6240 6241 6242 6243 6244 6245 Shake N Stack Oven 6240 200 2 B Rev 1 SHEET 2 OF 2 Page 1 of 2 8 8 Shake n Stack Thermo Scientific Section 8 Technical Specifications BILL OF MATERIALS PART NO PART DESCRIPTION 0
6. Scientific All rights reserved Shake n Stack Thermo Scientific Preface mportant operating and or maintenance instructions Read the accompanying text carefully Potential electrical hazards Only qualified persons should perform procedures associated vvith this symbol Equipment being maintained or serviced must be turned off and locked off to prevent possible injury Hot surface s present which may cause burns to unprotected skin or to materials which may be damaged by elevated temperatures Marking of electrical and electronic equipment which applies to electrical and electronic equipment falling under the Directive 2002 96 EC WEEE and the equipment that has been put on the market after 13 August 2005 This product is required to comply with the European Union s Waste Electrical amp Electronic Equipment WEEE Directive 2002 96 EC It is marked with the WEEE symbol Thermo Fisher Scientific has contracted with one or more recycling disposal companies in each EU Member State European Country and this product should be disposed of or recycled through them Further information on Thermo s compliance with this directive the recyclers in your country and information on Thermo Scientific products will be available at www thermofisher com Always use the proper protective equipment clothing gloves goggles etc Always dissipate extreme cold or heat and wear protective clothing Always follow good hygiene practices Ea
7. aus 9 lu jay 0 Apeai 51 331440 Sage ouuau 830 INO sjonpoJd JO sso JO sjijod 180 0 D nbl l sabewep onelu novu Burpnjour sabewep lenu nb suo Jo 109 1 Aue 10 9101 eq JOU jeys ouueu AlddV TIVHS 380080 YVINIILYWd v SSINLH 11118 111 83 40 S3ILNVHHVM ON 03110 1 1 13 1111 83111311 S3ILNVHSVM H3HLO 11 30 0311 NI ANY 3 01003 SI ALNVHHVM SIHL 00810 s p g04 peddius ae syed yuawasejdas pue pred 8667800 lin l s 514 ouueu 0 paunjaJ eq ysnwW sued Buruuojuoo uou ile uodo s ouuau 1v jueuidinbe 10 sjuauodujoo Aue Jo Um JO e oJdde 1011d a 16 ysnwW JUaWHedag 8801 188 65 0 2U ponad 70616 eurBluo y puo aq yed 0810 OdWOI aui 0 10 queuudinbe y 1 0 AJUeEM y puajxe JOU jjeys Ajueuew siy Jepun 1 tidinb 10 syed syusuoduioo Jo ired i Jo 3ueuig3e da NU amp JIeM SI WO papnjoxa ae sjoxseb pue 5 91 1 sse 6 sway ajqepuadx3 suledas Aue jo eoueuuojied 0 101d uon3eJip pue uoneuruuasjap AUeJJeM 10 p9 951002 aq 19 1ueunjede 592 201 29 eu JUaWaaIHe Jup Up siy Aq paJaAo9 JOU 8 6 uonejqijeo pue uonej eisu papnjoul 4096 asuadxa s ouuau 1e peoejdaJ 10 peJredai aq diusueunjo JO sjenazew Buruuojuo9 uou aq 0 0 sued zuauodwos AA S11 34 60 ponad 0 4894 3811 84 Buunp saumo 1uanbasqns Aue o spuayxe 7087000 up Up ou 0818 180
8. efficient processing of all types of blot The equipment is invaluable for every stage of the nucleic acid immobilisation and detection procedure from the initial electrophoretic separation through to vacuum transfer and finally Hybridisation and washing The procedures outlined in this booklet are intended as general guidelines for the use of Thermo Hybridisation Ovens and may not be ideally suited for every research application Thermo Scientific May 2003 Issue 7 For further information the user is recommended to refer to the follovving laboratory manuals in addition to literature cited in the references Fritsch J amp Maniatis T 1989 Molecular Cloning A Laboratory Manual 2nd Edition Sambrook Cold Spring Harbour Laboratory Press Edited by Hames B D amp Higgins S J 1985 Nucleic Acid Hybridisation A Practical Approach IRL Press Oxford Thermo Scientific May 2003 Issue 7 CHAPTER 2 HYBRIDISATION GUIDE NUCLEIC ACID TRANSFER PROCEDURES Dot Slot Blotting 1 10 DNA samples prepared from cell lysate must first be denatured to form single stranded DNA Heat to 100 C and incubate for 5 minutes Chill DNA samples on ice for 5 minutes Cut two pieces of Whatman 3MM filter paper and one sheet of membrane to the size of the Dot blotting unit Pre wet the membrane and filter paper in 6 x SSPE SSC then place in the unit and secure the lid If samples are to be spotted on manually pre we
9. hybridisation probes from plasmids containing a bacteriophage SP1 promoter Nucleic Acids Res 12 7035 Lathe R 1985 Synthetic Oligonucleotide probes deduced from amino acid sequence data Theoretical and Practical Considerations J Mol Biol 183 1 12 Wallace R B Schaffer J Murphy R F Bonner J Hirose T and ltakura K 1979 Hybridisation of synthetic oligodeoxyribonucleotides to 174 DNA the effect of single base pair mismatch Nucleic Acids Res 6 3543 Wood Wl Gitschier J Lasky L A and Lawn R M 1985 Base composition independent hybridisation in tetramethylammonium chloride A method for oligonucleotide screening of highly complex gene libraries Proc Natl Acad Sci 82 1585 Thermo Scientific May 2003 Issue 7 37 Thermo Fisher Scientific 401 Millcreek Road Marietta hio 45750 United States www thermofisher com
10. installation calibration procedures 8 2 Shake n Stack Thermo Scientific Section 8 Technical Specifications Additional Rotisseries Accessories 1 4 in 35mm Rotisserie P N 222032 Holds up to 10 long medium amp short bottles 2 76 in 70mm Rotisserie P N 222033 Holds up to 4 extra long bottles Rotissieries to fit the Shake n Stack and Maxi 14 Hybridisation Ovens P N 222042 Holds up to 0 8 x 05 fl oz 25 x 15ml tubes amp 1 0 x 1 7 fl oz 30 x 50ml tubes Rotisserie to fit the Shake n Stack and Maxi 14 Hybridisation Ovens 7 P N 222043 Holds up to 1 7 x 0 5 fl oz 50 x 15ml tubes amp 0 54 x 1 7 fl oz 16 x 50ml tubes Rotisserie to fit the Shake n Stack and Maxi 14 Hybridisation Ovens OA E Biot s s een P N 222044 Holds up to 1 5 x 1 7 fl oz 44 x 50ml tubes Shaking Platform P N 222000 Hybridisation Bottles Extra long bottle 2 8 x 11 8 in 70 x 300mm 8212177 P N 110094 Long bottle 1 4 x 11 8 in 35 x 300mm 5605077777 P N 110113 Medium bottle 1 4 x 9 8 in 35 x 250mm 550771 P N 110115 Short bottle 1 4 x 5 9 in 355 x 150mm 0020000 P N 110116 Bottle gripper 11 8 in 300mm for extra long bottles M 7 7 P N 222055 Bottle gripper 11 8 in 300mm for long medium bottles 2227777 P N 222056 Bottle gripper 5 9 in 150mm for short bottles 0205000 x
11. pre hybridisation and hybridisation solutions e g Denhardt s reagent and salmon sperm DNA An adequate pre hybridisation is important to block non specific hybridisation to the membrane Hybridisation and or washing conditions not stringent enough i Decrease salt concentration ii Increase temperature iii Increase concentration of SDS iv Increase wash times Membranes drying out This may often be the cause of an apparent overlap problem and may result from i Too low a probe volume ii Too slow a change over of solutions particularly when bulk processing iii Oven not level iv Excessive variable axis angle Residual agarose on membranes may cause foggy backgrounds Membranes should be rinsed in 2 x SSC to remove residual agarose and excess salt after blotting and prior to fixing especially following vacuum blotting Multiple filters not separated by mesh in bottles Autoradiography problems Random black spots and lightening flash markings on autoradiographs may be due to static electricity Shake n Stack A 3 Section 11 Appendix Factors Resulting in Signal Lower than Expected A 4 Shake n Stack Insufficient exposure time of membrane to film during autoradiography Inefficient transfer and or binding of nucleic acids to the nylon membrane Target sequence present at very low copy numbers Increase the amount of sample loaded on to the gel Probe sequence not present
12. required time period Step 5 should be repeated for any subsequent wash The washing protocol using the shaking platform will be identical to that used in the bottles Note Refer to Appendix I for additional information on Washing In addition to bulk processing of blots during stringency washing steps the shaking platform is ideal for performing several other stages of the blot generation and processing procedures Three examples are given as follows 1 Pre treatment of Gels prior to Nucleic Acid Transfer Gentle agitation of the gel during pre treatment steps is essential to prevent damage to the gel Pre treatment steps are usually performed at ambient temperature i Depurination 0 25M HCI 10 minutes ii Denaturation 1 5M NaCI 0 5M NaOH 30 minutes iii Neutralisation 1 5M NaCI 0 5M Tris CI pH 7 2 30 minutes Pre washing of Filters prior to Colony Blot Hybridisation Pre washing is often advantageous to remove colony debris and thus reduce background noise when screening bacterial colonies by hybridisation After lysing the bacterial colonies and fixing the DNA on the membrane pre wash in a solution of 2 x SSC 0 1 SDS at 65 C Use several changes of buffer After several washes residual colony debris may be removed by gently rubbing filter with a gloved finger Shake n Stack 4 5 Section 4 Methodology of Hybridisation 3 Probe Stripping from Filters after Hybridisation An example of probe stripping pro
13. rotisserie shaker switch cannot be operated without first turning on the power switch To the left of the rotisserie shaker switch is a dial to adjust the rotisserie speed from 5 15 rpm and the shaker speed from 4 10 rpm Instructions to set up the shaking platform are in Section 4 The temperature controller has 4 buttons along the lower edge of the controller When the power is switched on the display shows three dashes as the controller goes through its internal self tests which are completed within 5 seconds The display then shows the compensated temperature of the oven Figure 2 1 Temp Controller To set the controller temperature press and release the L1 button The L1 LED will blink on and off and the display will show 1SP for 1 second then the present setpoint value Press the up or down arrow button to change the desired setpoint within the oven operating range of 35 C to 85 C Press the Enter button to store the new setpoint value or press the X button to return to normal mode without saving the new setpoint value The warm up time for the oven varies according to the load and the ambient temperature When first switching on the oven no bottles installed allow approximately one hour for the temperature to stabilise Solutions should be pre warmed to the required temperature to minimise sample stabilisation time Thermo Scientific Section 2 Unpacking and Installation Set the Required For ease of
14. solution should be maintained between pH 5 9 Tetramethyl Ammonium Chloride TMAC Generally speaking A T base pairs are less stable than G C base pairs TMAC ata concentration of 3M shifts the melting temperature of A T base pairs to that of G C base pairs Thus it may be used in oligonucleotide Hybridisation so that the stringency of Hybridisation becomes a function of the probe length alone rather than probe length and G C content Thermo Scientific May 2003 Issue 7 34 APPENDIX Ill HYBRIDISATION BOTTLE SAFETY Use of the Bottle Gripper Catalogue No HB OV BGS to fit 15cm small bottles Catalogue No HB OV BGL to fit 30cm medium amp large bottles Catalogue No HB OV BGXL to fit 30cm extra large bottles This silicone rubber safety sleeve fits neatly over all our standard bottles and is included with every Thermo Oven we ship Helping you to safely tighten and unscrew caps e Making handling of hot bottles easier For more details and ordering information contact Thermo Scientific or your local Distributor Bottle Care Hybridisation Bottles are made of thick walled borosilicate glass which protects users from radiation and has excellent long term reliability M itis 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 p
15. to the bottle Then roll the bottle in order to unwind the mesh and membrane Continue until the membrane and mesh is fully unwound The mesh ensures the probe will have access to all parts of the Hybridisation membrane including those parts of the membrane which overlap Thermo Scientific May 2003 Issue 7 Figure 3 Inserting Membranes in a Bottle 1 Shallow tray containing 2 x SSPE SSC Mesh Membrane Roll up mesh amp membrane 2 Place 10 15mls of SSPE SSC into a bottle and then insert the roll centrally 3 Secure cap and holding bottle N horizontally roll to catch the trailing 09 edge of the mesh continue rolling in ge e the same direction until coil of mesh and membrane is vvell positioned 4 Pour out SSPE SSC and replace with pre warmed prehybridisation buffer 5 Place the bottle in the Oven so that it rotates in the Oven in the same direction as it was unrolled in step 3 Thermo Scientific May 2003 Issue 7 14 Prehybridisation 1 Once the membrane is in place in the bottle the SSPE SSC can be discarded and replaced vvith prehybridisation buffer This is easily done by removing the cap pouring off the SSPE SSC and then pouring in the prehybridisation buffer All the solutions used during Hybridisation should be pre warmed before use 10 20ml solution is recommended for medium bottles and 5 10ml for small bottles Higher volumes will be required if there is
16. use and reproducibility when hybridising in bottles the p ty y 8 Temp continued temperature display is calibrated to show internal bottle temperature as P opposed to oven air temperature VVhen the shaking platform is used allowance must be made for the difference between the temperature shown on the display and the temperature achieved within a liquid filled vessel on the shaker This difference can be calculated by filling the protocol specific vessel with enough distilled water to cover an immersible thermometer and setting the oven to the desired product temperature After enough time for the temperature to have stabilized record the thermometer reading and subtract this value from the setpoint value The remainder must be added to the setpoint value for execution of the actual protocol For instance a setpoint value of 55 C minus a thermometer reading of 58 C yields a remainder of 3 C which means that the adjusted setpoint value for this protocol will be 52 C Repeat this process to verify that the thermometer reading is within 1 C of the setpoint value This difference should be determined for each oven and or shaker protocol The lowest target temperature which the oven can achieve is 8 C above ambient temperature Avoid placing the unit in direct sunlight or in any area where it is likely to be exposed to hazardous or volatile chemicals When positioning the Hybridisation Oven ensure a gap of at least 2 in 5cm i
17. wash is not recommended and can cause background problems The final wash is a high stringency wash Use of a hand held monitor to give an indication of the decrease in radioactivity as the washes progress is recommended and should give some indication as to whether this final wash should be carried out In general terms the stringency of Hybridisation and washing steps is increased by increasing the temperature or by decreasing the salt concentration Hybridisation should be carried out under relatively low stringency conditions compared to the washing procedures It is generally simpler and more effective to adjust the stringency during the washing steps by altering the salt concentration rather than the temperature Probe Preparation The final probe concentration should be in the region of 25 50ng ml of Hybridisation solution at approximately 1 5 x 10866 The optimum length of probe is approximately 500 800bp Purification of the labelled probe to remove unreacted triphosphates will reduce background problems and is recommended for all Hybridisations Thermo Recovery kits are excellent for this purpose Probe solutions should be pre warmed to the Hybridisation temperature and care should be taken to ensure the membrane is not exposed to the concentrated probe solutions if adding it directly to the bottles Thermo Scientific May 2003 Issue 7 26 Summary of Factors Resulting in High Backgrounds 1 2 Hybridisation solutions
18. wash solution approx 100ml and replace the cap NOTE Room temperature washing or washing with solutions which have not been pre warmed may result in background problems see Chapter 7 page 27 Replace the bottle in the Oven and leave for the required time period If transferring from sandwich box or plastic bag methodology the times and temperatures are usually those which are currently being used approximately 20 minutes per wash step Repeat steps 1 3 for each additional wash The wash solutions temperatures etc should be those recommended by the membrane manufacturer or as detailed in Chapter 7 Washing Procedure NOTE All wash solutions should be pre warmed for best results Method 2 Washing in a Sandwich Box Some scientists prefer to remove the membranes from the bottles and wash them all in one container Washing may be performed very simply on the shaking platform in the Shake n Stack Midi Dual or Maxi 14 Ovens and is particularly useful when several membranes are being processed simultaneously ils 2 3 Remove the Hybridisation Bottle from the Oven Pour off the Hybridisation buffer Using a pair of forceps carefully remove the membranes and place them in a suitable container with a lid e g sandwich box or similar Using the forceps gently unroll the membranes Thermo Scientific May 2003 Issue 7 16 4 Add a sufficient volume of the pre vvarmed first wash solution to totally imme
19. 00120 FAN IMPELLER 2 14036 1 19MM x IMM 1D GROMMET 3 56129 24 VDC TRIDENT GEAR MOTOR 4 56130 FAN MOTOR 220V 5 10221 CAP 1 0UF 275V CLASS X 6 83021 IDLER PULLEY 1 83022 DRIVE PULLEY 8 222003 BUSHING 9 222010 DRIVE SHAFT 0 1222013 17 1 OVEN LINER ASSEMBLY 222013 32 5 MOTOR BRACKET 2 230206 FUSE 1 6A 5X20MM 2 3 350025 1 HEATER TO THERMAL CUTOUT CABLE 4 350025 2 MAINS INLET EARTH GROUND 5 350025 4 PCB TO HEATER CABLE 6 350025 5 PCB TO THERMAL SWITCH CABLE 350025 8 COVER EARTH LEAD 8 350025 8 COVER EARTH LEAD 9 400112 TEMPERATURE CUT OUT 20 460165 FILTERED INLET FUSED 21 590036 M3 X 6mm SS PHP SCREW 2 22 590042 Ma X 20mm SS PHP SCREW 6 23 590043 14 X 10mm SS PHP SCREW 24 610083 M3 SS HEX NUT 2 25 610084 194 SS HEX NUT 10 26 680021 3 X MIO SS PHP SCREW AB POINT 2 21 680024 46 32 X 3 8 SELF TAPPING SCREW 3 28 100044 M3 X 4MM LONG SS HEX SOCKET SET SCREW 29 100051 5 X 8MM LONG ALLOY STEEL HEX SOCKET SET SCREW 2 30 100058 SHAFT SUPPORT BEARING 2 31 730048 194 INTERNAL TOOTH LOCK WASHER 23 32 130091 M4 SS FLAT WASHER 22 33 130092 M3 SS FLAT WASHER 5 34 130094 M3 INTERNAL TOOTH LOCK WASHER 2 35 130095 M8 RED FIBER WASHER 2 36 800049 DRIVE BELT Thermo Scientific Exploded Drawing Model 6240 6241 6242 6243 6244 6245 Shake N Stack Oven 6240 200 2 B Rev 1 Page 2 of 2 Shak
20. 27 6g 7 49 Dissolve in 800ml of H20 and adjust pH to 7 4 with NaOH solution Adjust the volume to 1 litre with H2O and sterilise by autoclaving 3 100 x Denhardt s Reagent Ficoll 2g Polyvinyl pyrollidone 2g Bovine serum albumin Fraction V 2g Dissolve in 100ml HO filter and store in aliquots at 20 C 4 Salmon Sperm DNA Dissolve the DNA at a concentration of 1mg ml in sterile H2O sonicate to a length of 200 500bp and then stand in a boiling water bath for 10 minutes Aliquot and store at 20 C 5 Denaturing Solution NaCl 1 5M NaOH 0 5M Sterilise by autoclaving Thermo Scientific May 2003 Issue 7 29 10 Neutralising Solutions NaCl 1 5M Tris Cl pH7 2 0 5M Na EDTA 0 001M Sterilise by autoclaving 10 Sodium Dodecyl Sulphate SDS Also known as sodium lauryl sulphate Formamide Formamide should be de ionised before use Add 5g of a mixed bed ion exchange resin e g Biorad AG501 to 100ml formamide and stir for 1 2 hours Store at 20 C Pre wash Solution 5x SSC 0 5 SDS 1mM Prehybridisation Buffer for Southern Blots Prepare as follows 10ml Solution Volume Final Concentration 20 x SSPE or SSC 6 x SSPE SSC 100 x Denhardt s 0 5ml 1 5 x Denhardt s reagent 10 SDS 0 5 SDS Add denatured salmon sperm DNA to 500 Thermo Scientific May 2003 Issue 7 30 11 Prehybridisation Buffer for Northern Blots Prepare as follows 10ml
21. DATE BY CAD APPO DESCRIPTION OF REVISION THIS DOCUMENT CONTAINS PROPRIETARY INFORMATION AND SUCH INFORMATION IS NOT TO BE DISCLOSED TO OTHERS FOR ANY PURPOSE NOR MODEL PART NAME 6240 6241 6242 6243 6244 amp 6245 SNS OVENS DWG TITLE ELECTRICAL SCHEMATIC Schematic Model 6240 6241 6242 6243 6244 6245 Thermo Scientific e er NES ec SPOKEN Stack Tven MATERIAL TS ELELTRUSTATIC ThermoFisher PAIN CLL 6240 70 0 D REV 3 SENSITIVE DEVICES SCIENTIFIC TOLERANCE UNLESS OTHERWISE SPECIFIED DRAWING NUMBER SIZE Po e 3 01 3 BOX 649 MARIETTA OHIO 45750 ANGLES DECIMAL AC 6240 70 0 D g Shake n Stack 9 3 Declaration of Conformity Manufacturers Name Thermo Fisher Scientific Asheville LLC Manufacturer s Address 401 Millcreek Road Marietta Ohio 45750 U S A Product Description Shake and Stack Ovens Product Designations 6240 6241 6242 6243 6244 6245 Year of Initial C Marking 2005 Affected Serial Numbers Release 3 Release Level REL shown on Serial Tag This product conforms to the following European Union Directive s EMC 2004 108 EC LVD 2006 95 EC This product conforms to the follovving Harmonized nternational and National Standards EMC LVD EN 61326 1 EN 61010 1 2 Edition EN 61000 3 2 UL 61010 1 2 Edition EN 61000 3 3 CSA C22 2 No 61010 1 2 Edition Date of Declaration Augu
22. ERE SWITCH SWITCH 15 360259 08 4 CHAMBER HEATER 250 WATT 240V 137023 E z VOLTAGE SELECTOR ZER SET FOR 230V 6102 TEMPERATURE B CONTROLLER i CON2 7 8 17 8 19 20 9 20 m TEMPERATURE CONTROL 21 231229 25 22 2 MEI TEMPERATURE SENSOR IK OHM 25 C 23 290201 24 25 26 27 28 29 3 2 31 32 33 34 Electrical Schenotic 33 Model 35 6240 6241 6242 6243 6244 6245 2 37 Shake N Stack Oven 38 6240 70 0 D REV 3 Page of 3 Thermo Scientific Shake n Stack 9 1 Section 9 Electrical Schematics 9 2 LINE CORD 430108 LINE FIL 460185 FUSES 6 3A TYPE T 2 R I D 230210 POWER SW 360258 ROTISSER 156129 RES 20 310 FAN 56 ROT STOR ER FLUSE HOLDER TCH OPST E SHAKER MOTOR 1 3K OHM SPEED CONTROL 301 10K OHM 86 MOTOR 31 SSERE SWITCH MODELS 6241 6243 6244 6245 110 VAC 50 60Hz IPH 2 3FLA 1 A 5 GRO Bu r o ao 29 s 5 e b M 1 N L MOTOR MAINS I CON 3B CONI 8 POT CONS EE EV S MAIN PAA 1 CONTROL FAN BDARD 181770 uU E MOTOR RE SWITCH 18 13 7 He e H HEATER THERMAL CUT OUT VOLTAGE SELECTOR OPEN e 100 C gt SET FOR 115V 400172 EMPERATLIRE CO
23. NTROLLER 002 181 19 20 arte 360259 HEA ER ELEMENT 250 WATT 37024 TEMPERATURE CONTROL 231229 TEMPERATURE SENSOR K OHM e 25 C 2980201 Electrical Schenotic Model 8240 6241 6242 6243 6244 6245 Shake N Stack Oven 6240 70 0 D REV 3 Page 2 of 3 Shake n Stack Thermo Scientific Section 9 Electrical Schematics LC NJ LJ LC LC LC LC LC CO N CT LC Electrical WIRE REFERENCE CHART NO SIZE COLOR 1 75MM BROWN 2 75 2 BLUE 75MM GRN YEL 38 75MM GRN YEL 4 75 BROW 5 75 BLUE 5 SMM BLAC 7 SMM RED 8 SMM GREE 9 N A RESISTOR LEAD O 5MM GREE 1 75MM BLUE 220V MODELS 1 75MM RED 110V MODELS 2 75MM BROW 2A 75MM RED 3 75MM RED 4 T5MM PURPLE 5 22MM YELLOW 6 22MM YELLOW 7 22MM BLAC 8 22MM BROW g 22MM RED 20 22MM ORANGE 21 SENSOR BROW 22 SENSOR BLUE 24 75MM BROW 25 75MM BLUE 3 0 388 1 08 13 111 GSN LOC CCS CHG TEMP CONT 231223 TD 231228 2 OV 292 05 02 08 JAS JAS LON 6240 6242 COUNTRY DEPENDENT CORDSET OV 181 12 18 03 JAS JAS LON CHANGE TEMP CONTROLLER REL 2 0 N A 9 25 03 Gy MSB RELEASED FOR PRODUCT ION REV ECN NO
24. Shake n Stack Hybridisation Oven Operating and Maintenance Manual 7026240 Rev 0 Visit us online to register your warranty Thermo www thermoscientific com warranty SCIENTIFIC Manual Numher 7026240 Preface Models covered by this manual Models 6240 6241 6242 6243 6244 6245 Voltage 220 110 220 ncludes Shaking platform drip tray rotisserie Shaking platform drip tray rotisserie Drip tray rotisserie Drip tray rotisserie Drip tray Shaking platform drip tray 0 25036 0V 368 8 16 11 New controller Release 3 CCS REV ECR ECN DATE DESCRIPTION By Thermo Scientific Shake n Stack i Preface CAUTION Contains Parts and Assemblies Susceptible to Damage by Electrostatic Discharge ESD Important Read this instruction manual Failure to read understand and follow the instructions in this manual may result in damage to the unit injury to operating personnel and poor equipment performance Caution All internal adjustments and maintenance must be performed by qualified service personnel Material in this manual is for information purposes only The contents and the product it describes are subject to change without notice Thermo Fisher Scientific makes no representations or warranties with respect to this manual In no event shall Thermo be held liable for any damages direct or incidental arising out of or related to the use of this manual 2011 Thermo Fisher
25. age should the membrane be allowed to dry out Removal of the probe on the membrane may be carried out by boiling the membrane in 0 1 x SSPE SSC 0 196 SDS for 5 minutes Check for residual probe activity by autoradiography before hybridising again Thermo Scientific May 2003 Issue 7 10 Northern Blot RNA Hybridisation 1 Prehybridisation is carried out by incubating the membrane in Northern Blot Prehybridisation Buffer recipe on page 33 Denature salmon sperm DNA by boiling for 5 minutes and then chilling on ice Add the denatured salmon sperm DNA to the buffer to a final concentration of 100ug ml Incubate with agitation or in a rotisserie for at least one hour at 42 C The volume of prehybridisation buffer required varies according to he Hybridisation system being utilised In general terms the minimum volume of buffer should be used such that the membrane is covered by the fluid at all times approximately 0 1ml cm or if in a Hybridisation bottle 10 20ml for a large bottle and 5 10ml for a small bottle Denature the labelled probe by heating to 100 C and incubating for 5 minutes Chill on ice and add to the prehybridisation solution Depending upon the system utilised the probe may be added directly or alternatively some prehybridisation buffer is removed the probe added to this and then the solution replaced in the Hybridisation vessel Some researchers may prefer to use fresh Hybridisation solution Hybridise w
26. and or wash solutions not pre warmed before use Probe concentrations too high or probe not denatured When transferring Hybridisation protocols to bottles the volumes will be reduced Ensure that probe concentrations are adjusted accordingly Unincorporated nucleotides not removed from probe solution Insufficient prehybridisation or blocking agents in prehybridisation and Hybridisation solutions e g Denhardt s reagent and salmon sperm DNA An adequate prehybridisation is important to block non specific Hybridisation to the membrane Hybridisation and or washing conditions not stringent enough i Decrease salt concentration ii Increase temperature Increase concentration of SDS iv Increase wash times Membranes drying out This may often be the cause of an apparent overlap problem and may result from i Too low a probe volume ii Too slow a change over of solutions particularly when bulk processing Oven not level iv Excessive variable axis angle Residual agarose on membranes may cause foggy backgrounds Membranes should be rinsed in 2 x SSC to remove residual agarose and excess salt after blotting and prior to fixing especially following vacuum blotting Multiple filters not separated by mesh in bottles Autoradiography problems Random black spots and lightening flash markings on autoradiographs may be due to static electricity Thermo Scientific May 2003 Issue 7 27 Summ
27. ane is covered by the fluid at all times approximately 0 1ml cm or if in Hybridisation bottles 10ml for a large bottle and 5ml for a small bottle Thermo Scientific May 2003 Issue 7 4 Denature the purified labelled probe by heating to 100 C and incubating for 5 minutes then chill on ice and either add to the prehybridisation solution taking care not to pipette directly on to the membrane or dilute the probe in pre washed Hybridisation solution and use this to replace the prehybridisation solution 5 Hybridise with agitation or by rotating in bottles for approximately 12 hours at 65 C 6 Stringency washing steps are carried out as follows using large volumes at least 50ml of the following solutions which should be pre warmed to the required temperature 2x15 with 2 x SSPE SSC 0 1 505 at minutes 65 C 1x30 with 1 x SSPE SSC 0 1 505 at minutes 65 C 1x10 with 0 1 x SSPE 0 1 SDS at minutes SSC 65 C The final wash is a high stringency wash Use of a hand held monitor to give an indication of the decrease in radioactivity as the washes progress is recommended and should determine whether this final wash should be carried out See Appendix Il for further notes 7 Wrap the membrane in Saran Wrap and autoradiograph at 70 C in a cassette with an intensifying screen or use a phosphoimager Expose initially for approximately 12 hours or overnight 8 Ifthe membrane is to be reprobed at no st
28. antages over capillary blotting namely a Transfer is completed in 1 hour instead of at least 4 hours for capillary blotting or more usually overnight b Superior results are obtained with vacuum blotting Because of the rapid transfer time there is less lateral diffusion of the DNA during transfer to the Hybridisation membrane This results in sharper bands on autoradiographs Excellent blots may be prepared quickly and conveniently using the Thermo Vacu Aid apparatus Capillary Blotting 1 Capillary blotting requires a buffer reservoir containing 10 20 x SSPE SSC e g a bioassay dish with a raised support for the gel e g an inverted sandwich box Two sheets of Whatman 3MM filter paper cut to the same width and slightly longer than the gel should be pre wet in 10 20 SSPE SSC and placed across the raised support so that the ends are submerged in the buffer reservoir This serves as a wick for the flow of the buffer through the gel see Figure 2 2 Capillary Blot Assembly 2 Invert the gel and place centrally on the filter paper Any filter paper exposed at the edge of the gel should be covered in Saran Wrap used X ray film or similar to prevent flow of buffer around the edge of the gel Thermo Scientific May 2003 Issue 7 3 Place a sheet of Hybridisation membrane cut to the size of the gel and marked for orientation on to the surface of the gel taking care to avoid air bubbles The membrane should only be ha
29. ary of Factors Resulting in a Signal Lower than Expected 1 Insufficient exposure time of membrane to film during autoradiography or phosphoimaging Inefficient transfer and or binding of nucleic acids to the nylon membrane Target sequence present at very low copy numbers Increase the amount of sample loaded on to the gel Probe sequence not present in sufficient quantities Increase the concentration of probe or include 10 dextran sulphate which reduces the solvent volume and has the same effect No probe homology Double stranded DNA probe was not denatured see standard protocols Alternatively probe degraded This is more likely to occur when using RNA probes The specific activity of the probe was too low Consider factors such as the probe concentration during the labelling reaction half life of radio labelled triphosphates etc Hybridisation and or washing conditions were too stringent i Increase salt concentration ii Decrease temperature iii Reduce concentration of SDS iv Reduce wash times The Hybridisation time was too short Thermo Scientific May 2003 Issue 7 28 APPENDIX I SOLUTIONS FOR NUCLEIC ACID BLOTTING amp HYBRIDISATION PROCEDURES 1 20 x SSC 1 litre NaCl 175g Sodium citrate 88g Dissolve in 800ml of H2O and adjust pH to 7 0 with NaOH solution Adjust the volume to 1 litre with H2O and sterilise by autoclaving 2 20x SSPE 1 litre NaCl 175g NaH PO H2O
30. ase to add a labelled phosphate group to the 5 terminus The various protocols of radio labelling nucleic acid probes are widely available in the published literature e g Maniatis et a In addition there are several commercially available kits containing all the components required for the different types of labelling experiments High quality HPLC purified oligonucleotides are available online from Thermo Scientific visit our web site at www thermo com molecularbiology Thermo Scientific May 2003 Issue 7 19 CHAPTER 5 HYBRIDISATION GUIDE NON RADIOACTIVE SYSTEM The use of non radioactive labelling techniques is becoming increasingly important for nucleic acid Hybridisation There are currently a number of systems available Most are based on the labelling of probes with either biotin or the hapten Digoxygenin DIG Biotinylated probes are hybridised and then linked to an appropriate enzyme such as alkaline phosphatase Visualisation is achieved by reaction of the enzyme with a chromogenic substrate Similarly DIG labelled probes are detected by high affinity Fab fragments coupled to alkaline phosphatase Visualisation is achieved using chromogenic substrates such as NBT and BCIP which produces a dark blue colour Although both of these systems offer advantages over radio labelled probes no safety risk or stability problems they have been recognised as less sensitive Recently the sensitivity of detection has been in
31. ashing step to remove colony debris and hence reduce background noise Thermo Scientific May 2003 Issue 7 Pre vvashing is carried out as required by incubating in at least 200ml of prewash solution see Appendix for at least 30 minutes at 50 C Longer incubations and several changes of buffer will assist in pre washing When the colonies are sufficiently broken up residual colony debris may be removed by gently rubbing the colonies with a gloved finger This procedure is particularly advantageous when screening with oligonucleotides Plaque Blotting The above procedure with some minor modifications may also be utilised for the screening of bacteriophage plaques as follows 1 Aliquots of the bacteriophage stock should be mixed with plating cells and plated in soft agarose Incubate at 37 C until plaques are approximately 0 2 0 5mm in diameter approx 10 12 hours Chill the plates for about 1 hour to set the top agarose Place a Hybridisation membrane cut to size or pre cut disc on to the surface of the agarose and leave for at least 30 seconds Orientation points should be marked with a sterile needle Further replicas may be prepared by leaving the Hybridisation membrane for progressively longer periods of time on the surface of the agarose To process membranes further proceed as from stage number 5 as for Colony Blotting Pre washing the filters stage 9 should not be necessary Southern Blotting 1 Siz
32. biology Anumber of different techniques may be used to analyse target sequences from various sources These techniques are all variations on a theme which can be summarised according to the source of DNA or RNA as follows 1 Dot Slot Blot Hybridisation Asample of unfractionated DNA or RNA immobilised on a membrane 2 Colony Plaque Blot Hybridisation DNA derived from cultured bacteria directly or from bacteriophage plaques and immobilised on a membrane 3 Southern Northern Blot Hybridisation DNA or RNA which has been size fractionated by gel electrophoresis and then transferred to a membrane 4 Western Blot Hybridisation Western blotting refers to the detection of polypeptides blotted on to nitrocellulose with antibodies and is outside the scope of this manual In each case the basic principle remains the same The nucleic acid for analysis is immobilised on a solid support and then hybridised with a specific single stranded Hybridisation probe complementary in base sequence to the region of interest and labelled to facilitate its detection The solid support most widely used for Hybridisation reactions is the nylon Hybridisation membrane This offers several advantages over alternative matrices such as nitrocellulose most notably high physical strength and binding capacity The DNA is immobilised on the support by baking or UV cross linking The Thermo range of Hybridisation equipment offers a complete system for the safest and most
33. brane in Saran Wrap and autoradiograph at 70 C in a cassette with an intensifying screen Expose initially for approximately 12 hours or overnight Background Hybridisation problems are much more common when using oligonucleotide probes For this reason procedures to reduce background such as pre washing of colony filters Chapter 2 Colony Plaque Blotting and purification of the oligonucleotide to remove unincorporated nucleotides are particularly advantageous Thermo Scientific May 2003 Issue 7 24 CHAPTER 7 HYBRIDISATION GUIDE TROUBLESHOOTING GUIDE FOR NUCLEIC ACID HYBRIDISATIONS Our bottle system is intrinsically simpler and safer to use than other methods of Hybridisation such as Hybridisation in bags or plastic boxes In Hybridisation Ovens the temperature of the solutions is precisely controlled and regulated and in addition the rotisserie device ensures that the solutions are continuously and evenly distributed over the membrane Thus the optimum conditions for Hybridisation and washing are maintained throughout when using this system However during the transfer of protocols from bags to bottles some minor adjustments to the protocols may be necessary When loading the mesh and membranes into the bottles air bubbles should be avoided Ensure that the Oven is sited on a flat surface so that the probe solution is distributed evenly along the length of the bottles and that there is sufficient probe solution to cover t
34. branes in a Bottle 1 Place the bottles to be used in the Oven during the warm up period approximately 1 hour 2 Selecta piece of support mesh appropriate for the size of the membrane The recommended Hybridisation mesh is supplied by Thermo 3 Pre wet the mesh and Hybridisation membrane in a suitable tray containing 2 x SSPE SSC see Figure 3 1 4 Ensuring that the Hybridisation membrane exactly overlays the mesh roll both up into a tight roll If more than one membrane is to be hybridised in a bottle simply overlay further meshes and membranes as required before rolling It is important that each membrane is separated from any other by a piece of mesh Up to five 20 x 20mm membranes can be hybridised in a single Hybridisation bottle Thermo Scientific May 2003 Issue 7 12 Place 10 15mls SSPE SSC into a Hybridisation Bottle and then insert the roll in such a way that the leading edge inside the roll and the trailing edge are positioned relative to each other as shown in Figure 3 2 Place the bottle on a flat surface and then slowly unwind the membrane and mesh around the inside of the bottle by rocking and gently rolling the bottle along the surface No air bubbles should be visible between the membrane and the bottle If bubbles are present the membrane should be removed and re rolled The procedure should then be repeated more gently Rock the bottle backwards and forwards to attach the first part of the membrane
35. cation concentration enables the DNA strands to come together more readily thus reducing the stringency of Hybridisation EDTA is sometimes added to chelate magnesium ions which can have an unpredictable effect on Hybridisation reactions Thermo Scientific May 2003 Issue 7 33 Base Composition Three hydrogen bonds are formed during G C base pairing G C rich probes bind more efficiently but may tend to bind non specifically Therefore probes should have a G C content of approximately 50 60 Formamide Formamide acts as a destabilising agent which reduces hydrogen bond formation Hybridisation reactions using formamide are therefore performed at lower temperatures Probe Length amp Complexity A long complex probe will hybridise more slowly than a short probe High Molecular Weight Polymers e g Dextran Sulphate Dextran sulphate acts as an accelerator In effect it excludes the probe itself from a large volume of the probe solution effectively increasing local probe concentration It also acts to some degree as a blocking agent Blocking Agents For example sonicated salmon sperm DNA Denhardt s reagent Blocking agents act as analogues to the probe binding to regions in the filter that the DNA would otherwise tend to bind to non specifically Detergents Sodium dodecyl sulphate has the effect of increasing the stringency of Hybridisation and can act as a blocking agent pH For optimum results the pH of the Hybridisation
36. cedure is as follows a Wash the filter at 65 C in 5mM Tris CI pH8 0 0 2mM EDTA 0 1 x Denhardt s reagent for 1 2 hours b Check for residual activity by autoradiography then re probe 4 6 Shake n Stack Thermo Scientific Thermo Scientific Variable Axis Rotisseries Section5 Rotisseries There are two kinds of rotisseries available variable axis and tube rotisseries Information follows The Shake n Stack is equipped with a variable axis rotisserie This allows the user to alter the angle at which bottles can be held in the rotisserie A small offset from horizontal of between 1 15 causes fluid to flow from end to end within the bottles during rotation see Figure 5 1 This wave motion provides a more vigorous and active hybridisation or washing stage and can allow probe volumes to be reduced to as low as 0 07 0 17 fl oz 2 5ml To achieve the optimum active wave conditions requires adjustment of the rotisserie angle Note that a large angle offset from horizontal 15 combined with a low probe volume lt 0 03 0 07 fl oz 1 2ml can produce dry areas on the membrane and so should be avoided To alter the bottle angle 1 Release the two securing screws visible on the front rotisserie wheel using a Phillips screwdriver 2 Put the rotisserie into the oven and place a bottle in the rotisserie 3 Rotate the free wheel to the desired angle 4 Remove rotisserie and tighten the screw in the new posi
37. ch individual is responsible for his or her own safety Thermo Scientific Shake n Stack iii Preface Do You Need nformation or Assistance on Thermo Scientific Products If you do please contact us 8 00 a m to 6 00 p m Eastern Time at 1 740 373 4763 Direct 1 800 438 4851 Toll Free U S and Canada 1 877 213 8051 FAX http www thermoscientific com Internet Worldwide Web Home Page service led marietta thermofisher com Service E Mail Address Our Sales Support staff can provide information on pricing and give you quotations We can take your order and provide delivery information on major equipment items or make arrangements to have your local sales representative contact you Our products are listed on the Internet and we can be contacted through our Internet home page Our Service Support staff can supply technical information about proper setup operation or troubleshooting of your equipment We can fill your needs for spare or replacement parts or provide you with on site service We can also provide you with a quotation on our Extended Warranty for your Thermo Scientific products Whatever Thermo Scientific products you need or use we will be happy to discuss your applications If you are experiencing technical problems working together we will help you locate the problem and chances are correct it yourself over the telephone without a service call When more extensive service is necessary we will assist you with
38. creased significantly down to femtogram levels by using a chemiluminescent reaction The Boehringer DIG system with the same alkaline phosphatase conjugate will generate light with the chemiluminescent substrate AMPPD The resultant signals produced can be detected using standard X ray film or a phosphoimager in as little as 30 minutes Light continues to be given off for up to 48 hours for longer developments The DIG System in Thermo Hybridisation Ovens The small probe volumes even mixing accurate temperature control and ease of handling associated with Thermo Oven systems are equally suited to the success of non radioactive Hybridisation The protocol detailed below has been developed in conjunction with the Boehringer DIG system The prehybridisation Hybridisation and washing stages of the protocol using the DIG labelled probe are exactly as for radio labelled probes The subsequent wash stages associated with detection and visualisation of the probe is often performed in sandwich boxes but will work equally well in bottles This protocol has been optimised for DNA RFLP analysis It may require minor alteration to be used for other applications The following protocol applies to a 20cm x 20cm membrane Thermo Scientific May 2003 Issue 7 20 Electrophoresis amp Transfer Electrophorese and transfer DNA fragments on to positively charged Hybridisation membrane Boehringer Mannheim Cat N 1209 272 Bake at 120 C for 30 mi
39. crew the cap 3 Ifthe same buffer is to be used for hybridisation as for pre hybridisation simply pipette the probe into the pre hybridisation fluid in the bottle Take care to avoid pipetting the probe directly on to the membrane as this will result in hot spots Alternatively dilute the probe in pre warmed hybridisation solution outside the bottle Pour off the pre hybridisation solution and replace with the probe solution 4 Replace the cap and gently agitate the bottle to ensure an even distribution of the probe in the hybridisation solution 5 Place the bottle back in the Hybridisation Oven switch on the rotisserie and leave it to hybridise for the required time period Thermo Scientific Shake n Stack 4 3 Section 4 Methodology of Hybridisation VVash i ng Two methods for washing follow Washing in Hybridisation 1 Remove the bottle from the Oven Bottles 2 Pour off the hybridisation fluid and then half fill the bottle with the first pre warmed wash solution approx 3 38 fl oz 100ml and replace the cap Note Room temperature washing or washing with solutions which have not been pre warmed may result in background problems see Appendix I of the Hybridisation Guide at the end of this manual 3 Replace the bottle in the Oven and leave for the required time period If transferring from sandwich box or plastic bag methodology the times and temperatures are usually those currently being used approximately 20 mi
40. cy of hybridisation and washing steps is increased by increasing the temperature or by decreasing the salt concentration Hybridisation should be carried out under relatively low stringency conditions compared to the washing procedures It is generally simpler and more effective to adjust the stringency during the washing steps by altering the salt concentration rather than the temperature Probe Preparation The final probe concentration should be in the region of 25 50ng ml of hybridisation solution at approximately 1 5 x 106cpm ml The optimum length of probe is approximately 500 800bp Purification of the labelled probe to remove unreacted triphosphates will reduce background problems and is recommended for all hybridisations Probe solutions should be pre warmed to the hybridisation temperature and care should be taken to ensure the membrane is not exposed to the concentrated probe solutions if adding it directly to the bottles A 2 Shake n Stack Thermo Scientific Factors Resulting in High Backgrounds Thermo Scientific Section 11 Appendix Hybridisation solutions and or vvash solutions not pre vvarmed before use Probe concentrations too high or probe not denatured When transferring hybridisation protocols to bottles the volumes will be reduced Ensure that probe concentrations are adjusted accordingly Unincorporated nucleotides not removed from probe solution Insufficient pre hybridisation or blocking agents in
41. d method will not damage the equipment 6 2 Shake n Stack Thermo Scientific Thermo Scientific Section 7 Use of Radioactive Probes Thermo s Hybridisation Ovens have been tested with a variety of non radioactive probes e g the DIG Non Radioactive Nucleic Acid labelling and detection system from Boehringer Mannheim Please refer to the Hybridisation Guide for guidelines on the use of non radioactive systems Warning The Shake n Stack Oven has not been designed for use with hazardous or volatile chemicals with low flash points such as dimethylformamide or chloroform Thermo cannot accept responsibility for damages arising from such use If in any doubt contact a Thermo product specialist prior to use Shake n Stack 7 1 Thermo Scientific Section 8 Technical Specifications Temperature ana a npe M 35 C to 85 C Selection Saee Digital Display Digital Monitor Sensor 225 essa s Thermistor Uniformity in bottle 0 25 C Accuracy at 539 EE 1 0 C Safety Over Temp Cut Out 100 C Rotisserie Rotisserie Speed 2 ad v dud eu eA AS 5 15 rpm Capacity 10 long medium amp short bottles Material awesta Stainless steel shaft a Delrin plastic rotisserie vvheels WEE eive Da eset pU Ms Variable axis 0 15 Shaker Speed eRe 4 10 rpm Maximum Weight Capacity 2 2 lb 1kg Max Load Dimens
42. direct factory trained technicians or a qualified service organization for on the spot repair If your service need is covered by the warranty we will arrange for the unit to be repaired at our expense and to your satisfaction Regardless of your needs our professional telephone technicians are available to assist you Monday through Friday from 8 00 a m to 6 00 p m Eastern Time Please contact us by telephone or fax If you wish to write our mailing address is Thermo Fisher Scientific 401 Millcreek Road Box 649 Marietta OH 45750 International customers please contact your local Thermo Scientific distributor iv Shake n Stack Thermo Scientific Thermo Scientific Section 1 Section 2 Section 3 Section 4 Section 5 Table of Contents Introduction Oven Temperature Monitoring Safety Precautions Unpacking and Installation Operational Set the Required Temperature Set Up the Shaking Platform Methodology of Hybridisation Place Membranes in a Bottle Insert Membranes in a Bottle Pre Hybridisation Hybridisation Washing ere s d VVashing in Hybridisation Bottles VVashing in a Sandvvich Box RotisserieS Variable Axis Rotisseries 50ml amp 15ml Tube Rotisseries Shake n Stack Table of Contents Section 6 leaning and Decontam
43. disations are conveniently performed in the dedicated Hybridisation equipment available from Thermo The Hybridisation Oven range consists of incubators with integral rotisserie devices which can accommodate 4 10 or 14 Hybridisation bottles 35mm in diameter in order to perform Hybridisations in minimal probe volumes with fluid moving continually over the membrane Southern Blot DNA Hybridisations The following protocol is broadly applicable to Hybridisations using DNA probes of 50bp and above following transfer and fixing of DNA to membranes as described in Chapter 2 For Hybridisations using oligonucleotides refer to Chapter 6 The Hybridisation procedure consists of four stages 1 Prehybridisation 2 Hybridisation 3 Stringency washing 4 Autoradiography For detailed notes on Hybridisation specific to Thermo equipment refer to Notes for Nucleic Acid Hybridisation on page 11 1 Prehybridisation is carried out by incubating the membrane in Southern Blot Prehybridisation Buffer for recipe see page 30 Denature salmon sperm DNA by boiling for 5 minutes and then chilling on ice Add the denatured salmon sperm DNA to the buffer to a final concentration of 5000 2 Incubate with agitation or in a rotisserie for a least 1 hour at 65 C 3 The volume of prehybridisation buffer required varies according to the Hybridisation system being utilised In general terms the minimum volume of buffer should be used such that the membr
44. e fractionation of the DNA is carried out by agarose gel electrophoresis a suitable range of horizontal gel apparatus is available from Thermo Scientific Before transfer to Hybridisation membranes the DNA in the agarose must be treated to ensure efficient transfer and to generate single stranded DNA suitable for Hybridisation Gentle agitation of the gel is essential to prevent damage to the gel during these steps The shakers in Shake n Stack Midi Dual 14 and Maxi 14 are ideal for this purpose Depurination to break the DNA into smaller fragments suitable for transfer is recommended for transfer of all DNA fragments large than 10kb and may assist transfer of smaller fragments Place the gel in a solution of 0 25M HCI for 10 minutes at room temperature with gentle shaking Thermo Scientific May 2003 Issue 7 3 Replace the depurinating solution with denaturing solution Denaturation for at least 30 minutes is recommended for most applications with a change of solution after 15 minutes 4 Replace denaturing solution with neutralising solution and gently shake the gel for least 30 minutes replacing with fresh solution after 15 minutes 5 The DNA is then transferred to a Hybridisation membrane cut to the size of the gel At this stage a corner of the membrane should be cut to assist later in orientation Transfer may be carried out by capillary blotting or more efficiently by vacuum blotting Vacuum blotting offers significant adv
45. e n Stack 8 9 Section 8 Technical Specifications 8 10 PART OF T HANDLE BILL OF MATERIALS NG PART NO PART DESCRIPTION QTY 116120 DOOR HINGE 2 2 121076 SATIN SILVER T HANDLE 3 179011 DOOR WINDOW 4 222012 0008 MOULDING 5 1222013 33 1 D00R CLAMP FRAME 6 680020 l x 9 5mn PHP SCREW AB POINT 4 7 680022 8 X 1 2 SS FHP TYPE A SCREW 8 BACK GROUND WIR NG Exploded Drawing Model 6240 6241 6242 6243 6244 6245 Stack N Shake Oven 6240 200 3 B Rev 0 Page 1 of 1 Shake n Stack Thermo Scientific Section 9 Electrical Schematics MODELS 6240 6242 220 VAC 50 80Hz IPH 1 IFLA CN LINE CORD COUNTRY DEPENDENT 2 20 LINE FILTER FUSE HOLDER 3 1 a p 1 460185 244 2 FUSES 1 6A TYPE T 2 REDD ZE X io 230205 4 Y CAPACITOR 1 0 UF CLASS X2 GRO 170227 ees E POWER SWITCH OPST 360258 6 7 x ROTISSERIE SHAKER MOTOR 155128 8 9 3 RESISTOR 1 3K OHM 1201301 1 aka SPEED CONTROL 10K CHM 310188 MAIN CONTROL INTERIOR CHAMBER FAN 2 e BOARD 156130 191770 3 MOTOR ROTISS
46. e region of interest which may be radio labelled during the amplification reaction itself or the final product labelled post amplification Labelling of double stranded DNA may be carried out by NICK TRANSLATION OR PRIMER EXTENSION Commercially available kits enable these techniques to be carried out simply and efficiently resulting in probes of high specific activity often requiring no further purification before adding to the Hybridisation buffer Removal of unincorporated nucleotides may be advantageous in reducing background This may be simply carried out using Thermo Recovery kits Poo N Single Stranded DNA Probes Single stranded DNA probes may be synthesized from mRNA using Reverse Transcriptase or may be derived from fragments cloned into specialised M13 or phagemid vectors which contain the origin of replication of a single stranded DNA bacteriophage Synthesis of the DNA strand complementary to the region of interest incorporating a 2 labelled dNTP results in a single stranded radio labelled probe molecule which is then separated from the unlabelled template by gel electrophoresis the fragment may then be extracted from the gel using Thermo Recovery kits This technique eliminates the possibility of re association of complementary strands which can occur with double stranded DNA probes Thermo Scientific May 2003 Issue 7 18 RNA Probes RNA probes are also derived from fragments which have been cloned into special
47. es are designed to accept the long medium or short Hybridisation Bottles Once unpacked attach leveling feet to base and position on a flat surface Level the oven by adjusting the height of the leveling feet Once leveled the oven is ready for use If adding to a stack of Shake n Stack Ovens maximum of 3 attach feet and adjust height then position feet in the 4 indents on the top of the supporting Shake n Stack Oven Some additional adjustment of the feet may be necessary until the ovens sit in a stable position Note Shake n Stack Ovens have been designed to stack 3 units high No attempt should be made to stack higher than this Connect each individual oven to the mains supply using only the removable mains lead supplied The mains lead should be fitted with a 10A fuse for both 110 120V and 220 240V regions The oven is grounded via the mains lead and should only be connected to an grounded supply See Technical Specifications Shake n Stack 2 1 Section 2 Unpacking and Installation 2 2 Operation Set the Required Temperature Shake n Stack The two switches found on the control panel operate the power and the rotisserie drive The power switch is marked with a 1 and a 0 By switching to the 1 position the mains power is connected to the oven and the green section of the switch is exposed to indicate that the oven is ON The switch to the right operates the rotisserie shaker The
48. he entire membrane On occasions the mesh and membrane can become tightly rolled up in the bottle This occurs if the mesh is loaded incorrectly see Figure 3 in Chapter 3 Background Reduction General All solutions for nucleic acid Hybridisations should be prepared using distilled water and highest quality reagents in clean glassware In particular water with a high organic content will cause bad background problems Formamide should be freshly de ionised Membranes should always be handled wearing gloves or with forceps The following should also be taken into consideration Prehybridisation Procedure Prehybridisation is required to block the sites on the nylon membrane which the probe would otherwise bind to non specifically Failure to carry out adequate preHybridisation results in high backgrounds If dextran sulphate is used in the Hybridisation solution then it must also be included in the prehybridisation solution Thermo Scientific May 2003 Issue 7 25 V ashing Procedure Stringency vvashing should be carried out as follovvs using large volumes approximately 100ml of the following solutions which should be pre vvarmed to the required temperature 1 2 x 15 minutes with 2 x SSPE SSC 0 1 SDS at 65 C 2 1 x30 minutes with 2 x SSPE SSC 0 1 SDS at 65 C 3 1 x10 minutes with 0 1 x SSPE SSC 0 1 SDS at 65 C All wash solutions should be pre warmed to the appropriate temperature An initial room temperature
49. igh organic content will cause bad background problems Formamide should be freshly de ionised Membranes should always be handled wearing gloves or with forceps The following considerations should also be applied Pre Hybridisation Procedure Pre hybridisation is required to block the sites on the nylon membrane which the probe would otherwise bind to non specifically Failure to carry out adequate pre hybridisation results in high backgrounds If dextran sulphate is used in the hybridisation solution then it must also be included in the pre hybridisation solution Shake n Stack A 1 Section 11 Appendix Bac kq round Washing Procedure Reduction continued Stringency vvashing should be carried out using large volumes approximately 100ml of the following solutions which should be pre warmed to the required temperature 1 2 x 15 minutes with 2 x SSPE SSC 0 1 SDS at 65 C 2 1x 30 minutes with 2 x SSPE SSC 0 1 SDS at 65 C 3 1x 10 minutes with 0 1 x SSPE SSC 0 1 SDS at 65 C All wash solutions should be pre warmed to the appropriate temperature An initial room temperature wash is not recommended and can cause background problems The final wash is a high stringency wash Use of a hand held monitor to give an indication of the decrease in radioactivity as the wash progresses is recommended and should give some indication as to whether this final wash should be carried out In general terms the stringen
50. in sufficient quantities Increase the concentration of probe or include 10 dextran sulphate which reduces the solvent volume and has the same effect No probe homology Double stranded DNA probe was not denatured see standard protocols Alternatively probe degraded This is more likely to occur when using RNA probes The specific activity of the probe was too low Consider factors such as the probe concentration during the labelling reaction half life of radiolabelled triphosphates etc Hybridisation and or washing conditions were too stringent i Increase salt concentration ii Decrease temperature iii Reduce concentration of SDS iv Reduce wash times The hybridisation time was too short Thermo Scientific Thermo Scientific Bottle Care Appendix II Hybidisation Bottle Safety Use of the Bottle Gripper Part No 222057 fits 6 in 15cm short bottles Part No 222056 fits 12 in 30cm medium long bottles Part No 222055 fits 12 in 30cm extra long bottles This silicone rubber safety sleeve fits neatly over all our standard bottles and is included with every oven we ship e Helping you to safely tighten and unscrew caps e Making handling of hot bottles easier For more details and ordering information contact Technical Services or your local distributor Our Hybridisation Bottles are made of thick walled borosilicate glass which protects users from radiation and has excellent long
51. ination 6 1 ee ee edu 6 1 Bottles amp Caps uec peed Age ek E PO tre 6 2 Oven Intetior Shakine Platferm d 6 2 1 8818582716 D ras De bow Seen 6 2 Section 7 Use of Radioactive Probes 7 1 Section 8 Technical Specifications 8 1 Additional Accessories bit hove es da a 8 3 Exploded Parts Drawings ac suec dr a eae ee LEES 8 4 Section9 Electrical Schematics 9 1 Section 10 Warranty Information 10 1 Appendix 1 Troubleshooting Guide for Nucleic Acid Hybridisations Al 1 Background Reduction General AL Factors Resulting in High Backgrounds 1 3 Factors Resulting in Signal Lovver than Expected 1 4 Appendix II Hybidisation Bottle Safety All 1 Bottle Care sss ac koa EE ve 11 1 Shake n Stack Thermo Scientific Oven Temperature Thermo Scientific Monitoring Section 1 Introduction The Thermo system of Hybridising in bottles is considered by many molecular biologists to be the best method for conducting Hybridisations with Southern Northern Dot Slot or Colony Blots Additional information includ
52. ing detailed protocols is contained in the included Hybridisation Guide Hybridising in bottles means that probe volumes may be significantly reduced compared to experiments performed in conventional systems and the continual movement of the probe across the surface of the membrane results in very efficient hybridisation reactions Protection from exposure to radiation is provided by the heavy walled borosilicate glass bottles Warning Based on tests these bottles block almost 100 of BETA radiation emissions of a 1 000 pCi P 32 source However users should observe precautions due to induced X ray phenomenon Breemsstrahlung occurring depending on the type s of radionuclides used The Hybridisation Oven itself provides additional shielding In the event of a spillage within the Oven the stainless steel drip tray will contain up to 6 8 fl oz 200ml of liquid Note If the Shake n Stack is not used as specified in this manual the protection provided by the equipment may be impaired All Ovens are calibrated by matching the thermistor temperature read out seen on the LCD display to the actual measurement of distilled water contained within a Hybridisation Bottle as it rotates The temperature measuring device used inside the calibration bottle is traceable to national standards This calibration method ensures all experiments using the rotisserie meet our temperature accuracy specification of 1 C and indeed due to the ro
53. into either slot on drive shaft ensuring the sleeve is pushed fully onto shaft Tighten using thumb screw 4 Locate front left peg of tray into the hole at the end of the drive arm Figure 5 Tray Peg into Arm Hole 5 Slide the rear left peg into the horizontal slot on the left hand side of bracket at the back of oven Assembly is now complete and ready to operate Figure 6 Rear Peg into Slot 3 2 Shake n Stack Thermo Scientific Place Membranes in Thermo Scientific a Bottle Section 4 Methodology of Hybridisation The following procedures indicate how to use the Shake n Stack Oven for hybridisation Place the bottles to be used into the oven during the vvarm up period approximately 1 hour Select a piece of support mesh appropriate for the size of the membrane The recommended hybridisation mesh is supplied by Thermo see parts list in Section 8 Pre wet the mesh and hybridisation membrane in a suitable tray containing 2 x SSPE SSC See Figure 4 1 Ensuring that the hybridisation membrane exactly overlays the mesh roll both up into a tight roll If more than one membrane is to be hybridised in a bottle simply layer meshes and membranes as required before rolling It is important that each membrane is separated from another by a layer of mesh Up to five 0 8 x 0 8 in 20 x 20mm membranes can be hybridised in a single hybridisation bottle Place 0 34 0 51 fl oz 10 15ml SSPE SSC in
54. ions 9 8 VV x 7 1 H x 7 9 D s 250mmW x 180mmH x 200mmD NISteriab a pesci tea S Stainless steel Dimensions External 17 1 VV x 16 7 H x 14 3 D ESS 435mmW x 425mmH x 363mmD Internal 13 67 7 x 9 6 H x 9 5 D TOS MON 345mmW x 245mmH x 240mmD Shipping Weight 53 Ib 24kg Shake n Stack 8 1 Section 8 Technical Specifications Features Plastic Snap fit Rotisserie 577775 Easy Set Temperature Controller Standard Accessories Shaking Tray Drive Arm Assembly nin Models 6240 6241 6244 6245 2200000 0 Shaking Tray Bracket un Models 6240 6241 6244 6245 OPORTET Povver Cable 0000 7 1 4 in 35mm Rotisserie 00200 Models 6240 6241 6244 6245 Ordering Information Shake n Stack LION a ga en e Model 6241 2 20 ia pal d Model 6240 Includes shaking platform drip tray and Delrin plastic rotisserie KIEM Tt heo a cease eee Medline Model 6243 RAR Model 6242 Includes drip tray and Delrin plastic rotisserie E DC PEE Model 6245 Includes shaking platform and drip tray LIUM pu M Model 6244 Includes drip tray Accessory Pack P N 222060 Includes 2 medium bottles and Bottle Gripper Temp Controller Replacement Kit DIN 192048 Includes temperature controller PTC probe gasket and
55. is J Mol Biol 98 503 Medveczky P Chang C W Oste C and Mulder C 1987 Rapid vacuum driven transfer of DNA and RNA from gels to solid supports Biotechniques 5 242 Olszewska E and Jones K 1988 Vacuum blotting enhances nucleic acid transfer Trends Genet 4 92 Kroczek R A and Siebert E 1990 Optimisation of Northern Analysis by Vacuum Blotting RNA Transfer Visualisation and Ultraviolet Fixation Anal Biochem 184 90 Rigby P W J Dieckman MI Rhodes C and Berg P 1977 Labelling Deoxyribonucleic Acid to high specific activity in vitro by nick translation with DNA polymerase LJ Mol Biol 113 237 Thermo Scientific May 2003 Issue 7 36 10 11 12 13 14 15 16 17 Feinberg A P and Vogelstein B 1984 A technique for radio labelling DNA restriction endonuclease fragments to high specific activity Anal Biochem 132 6 Feinberg A P and Vogelstein B 1984 Addendum A technique for radio labelling DNA restriction endonuclease fragments to high specific activity Anal Biochem 137 266 Messing J 1983 New M13 vectors for cloning Methods Enzymol 101 20 Ley T J Anagnon M P Pepe G and Nienhius A W 1982 RNA processing errors in patients with B thalassimia Proc Natl Acad Sci 19 4775 Melton D A Krieg P A Rebagliati M R Maniatius T Zinn K and Green M R 1984 Efficient in vitro synthesis of biologically active RNA and RNA
56. ised cloning vectors These vectors contain the promoter sequences of a bacteriophage encoded DNA dependent RNA polymerase for example SP6 to T7 adjacent to a multiple cloning site into which the fragment is inserted Transcription of the cloned fragment in the presence of the specific RNA polymerase and a 5 P labelled rNTP results in labelled RNA The template DNA is removed simply by DNAse digest Vectors of this type are available containing two phage promoters in opposite orientations adjacent to the multiple cloning site so that the strand to be transcribed may be chosen according to the phage RNA polymerase used These RNA probes have become the probes of choice for many applications the only limitations being the additional care required for handling RNA Synthetic Oligonucleotides An oligonucleotide probe of a single defined DNA sequence may be synthesized if the target nucleic acid sequence is available using a DNA synthesis machine or commercial service Alternatively pools of oligonucleotides of degenerate sequences may be synthesized derived from an amino acid sequence of a gene product Due to the degeneracy of the genetic code i e most amino acids are specified by more than one triplet codon a given amino acid sequence will be coded for by a number of alternative DNA sequences one of which will represent the specific target sequence for the probe Oligonucleotides are most commonly radio labelled using T4 Polynucleotide Kin
57. ith agitation or rotating in bottles for approximately 12 hours at 42 C Stringency washing steps are carried out as follows using large volumes at least 50ml of the following solutions which should be pre warmed to the required temperature 2x15 with 2 x SSPE 0 1 SDS at minutes 42 C 1x30 with 1x SSPE 0 1 SDS at minutes 42 C 1x15 with 0 1 x 0 1 SDS at minutes SSPE 42 C Thermo Scientific May 2003 Issue 7 11 The final vvash is a high stringency vvash Use of a hand held monitor to give an indication of the decrease in radioactivity as the vvashes progress is recommended and vill determine if the final vvash should be carried out 7 Wrap the membrane in Saran Wrap and autoradiograph at 70 C in a cassette vvith an intensifying screen or use a phosphoimager Expose initially for 12 hours or overnight 8 Ifthe membrane is to be reprobed at no stage should the membrane be allowed to dry out Removal of the probe on the membrane may be carried out by washing the membrane for 1 2 hours at 65 C in 5mM Tris HCI pH8 0 0 2mM EDTA 0 1 x Denhardt s reagent Notes for Nucleic Acid Hybridisations using the Thermo Range of Equipment The Hybridisation Oven range has been designed to provide the optimum conditions for performing all types of Hybridisation and stringency washing procedures safely and simply Hybridisations are performed in bottles to maximise user safety and to minimise probe volumes Placing Mem
58. led Ajuguew EU BL O v puo eq ed jueuoduJoo y JO jueuudinbe y JOYE O Aju amp JJe y puejxe jou jeus 0 16 SIU spun jueuudinbe Jo sued sjueuoduloo edau JO 4 Queer SIU pepnjoxe sue sjeyxseD pue Buiqn sjueBeai suey ssej6 sway ejqepuedx3 s ede Aue jo eoueuuojied oj soud uonoeupp pue uoneuiuJejep AJUBUeM JO pejoejuoo eq snw jueunjede seoiueg 128214420 eu 62 Aue em siu Aq paJoAoo JOU uoneJgi eo pue uorejejsu pepnjoxe Joqe su dx s ouueu je peoe daiJ Jo peJredaJ eq dIYSUBLUYJOM JO s eruejeuu Ul Buruojuoo uou 2q oj USAOJd sued jueuoduioo 1511 eu 6 7080718080 60 88 E91uu99 v O peuunjeJ pue pejejduJoo si DIE uenem v pepi oJd uonej ejsur pue ajap JO syzuow xis jeuonippe ue D AQIIE aie jueuudinbe xoo1s oym sJe ee poled Jes si v Duunp saumo juenbesqns Aue o spua x2 uoi 99 01d 8 6 SU peJeAr ep SI jueuudinbe euin ewes v A ojyeuurxoudde we JOSS out of Aju amp JJeM y os euin Buiddius 104 Swoj e siu AuIDEL JNO wo peddius si jueuudinbe JnoA ejep Sy sujuoul OM SUEIS 09 uenem au ALNVSHHVM 3 1V3G 8 313111 3111113105 YAHSIA 0 381 A 7 Shake n Stack Thermo Scientific HYBRIDISATION GUIDE USER INSTRUCTION MANUAL Manual 7222060 Rev 0 HYBRIDISATION GUIDE Contents CHAPTER 1
59. mbrane is well positioned Figure 4 3 Rolling Direction of Bottle Figure 4 4 Bottle in Oven Rotation 4 Pour out SSPE SSC and replace with pre warmed pre hybridisation buffer 5 Place the bottle in the oven so that it rotates in the oven in the same direction as it was unrolled in Step 3 4 2 Shake n Stack Thermo Scientific Section 4 Methodology of Hybridisation Pre Hybridisation 1 Once the membrane is in place in the bottle the SSPE SSC can be discarded and replaced with pre hybridisation fluid This is easily done by removing the cap pouring off the SSPE SSC then pouring in the pre hybridisation fluid All the solutions used during hybridisation should be pre warmed before use 0 34 0 51 fl oz 10 20ml solution is recommended for medium bottles and 0 17 0 34 fl oz 5 10ml for short bottles Higher volumes will be required if there is more than one membrane in the bottle e g 0 51 0 85 fl oz 15 25ml 2 Replace the cap on the bottle and insert into the rotisserie Hybridisation Denature the purified probe by boiling for five minutes then store on ice If the volume to be used for hybridisation is substantially different than that used previously for hybridisation in bags or boxes ensure that the quantity of the probe is adjusted accordingly to maintain the correct probe concentration If this is not done high background may result 2 Remove the bottle from the Hybridisation Oven and uns
60. more than one membrane in the bottle e g 15 25ml Replace the cap on the bottle and insert into the rotisserie Hybridisation 1 Denature the purified probe by boiling for 5 minutes then store on ice If the volume to be used for Hybridisation is substantially different to that used previously for Hybridisation in bags or boxes ensure that the quantity of the probe is adjusted accordingly to maintain the correct probe concentration If this is not done high background may result Remove the bottle from the Hybridisation Oven and unscrew the cap If the same buffer is to be used for Hybridisation as for prehybridisation simply pipette the probe into the prehybridisation fluid in the bottle Take care to avoid pipetting the probe directly on to the membrane as this will result in hot spots Alternatively dilute the probe in pre warmed Hybridisation buffer outside the bottle Pour off the prehybridisation buffer and replace with the probe solution Replace the cap and gently agitate the bottle to ensure an even distribution of the probe in the Hybridisation solution Place the bottle back in the Hybridisation Oven switch on the rotisserie and leave it to hybridise for the required time period Thermo Scientific May 2003 Issue 7 15 V ashing Method 1 Washing in Hybridisation Bottles 1 2 Remove the bottle from the Oven Pour off the Hybridisation buffer and then half fill the bottle with the first pre warmed
61. nated it will be necessary to leave it to decontaminate in Decon 90 for a longer time period 5 Meshes should be allowed to dry flat between uses Thermo Scientific Shake n Stack 6 1 Section 6 Cleaning and Decontamination Bottles amp Ca DS The hybridisation bottles and caps can be decontaminated by the following procedure 1 Soak the bottles and caps in a dilute detergent solution Decon 90 overnight 2 Remove the Decon 90 and rinse with distilled water 3 Ifthe bottles and caps have been decontaminated no further action is necessary If however they are still contaminated gently scrub them with an abrasive cloth or brush and if necessary leave to decontaminate in Decon 90 for a longer time period Oven Interior Shaking Stainless steel drip trays are provided with all Thermo ovens and are Platform intended to contain spillages in the event of an accident These together with the stainless steel surfaces of the ovens and the shaking platform can be decontaminated by wiping clean with Decon 90 followed by distilled water No further action should be necessary Rotisserie The rotisserie may be cleaned with Decon 90 and distilled water No further action should be necessary Avoid alcohol or other organic solutions which may affect the plastic surfaces Caution Before using any cleaning or decontamination method except those recommended by the manufacturer users should check with the manufacturer that the propose
62. ndled vvearing gloves or using forceps Then cover the membrane with two sheets of Whatman 3MM paper cut to just larger than the gel 4 Place a stack of absorbent paper towels on top of the filter paper weighted down with a 1kg weight The stack of towels should be approximately 5cm deep sufficient to continue drawing buffer through the gel until transfer is complete which takes at least 4 hours The stack of towels should also be level to ensure even transfer Take care to ensure that the stack of towels is not in contact with the buffer wick which would cause a short circuit of buffer bypassing the gel Usually it is convenient to transfer overnight 5 After blotting is complete the apparatus is dismantled At this stage it is possible to mark the position of the wells on the Hybridisation membrane with a soft pencil The membrane is then rinsed briefly in 2 x SSPE SSC and air dried on a sheet of dry filter paper 6 Fix the DNA to the Hybridisation membrane either by baking at 80 C for 2 hours or by UV cross linking 7 After fixing the DNA the membrane is ready for Hybridisation Membranes not hybridised immediately may be stored between sheets of Whatman 3MM paper in sealed plastic bags at 4 C Figure 2 1 Capillary Blot Assembly Weight Stack of paper Transfer solution reservoir 3MM paper ps Nylon membrane 277 Agarose gel eee 77 Gel support 3MM VVhatman filter paper vvick Tran
63. ntific May 2003 Issue 7 CHAPTER 7 TROUBLESHOOTING GUIDE FOR NUCLEIC ACID HYBRIDISATIONS USING THERMO OVENS 25 Background Reduction General 25 Prehybridisation Procedure esses 25 Washing Procedure cio on aaa 26 Probe Preparationiz soot ay d 26 Summary of Factors Resulting in High Backgrounds 27 Summary of Factors Resulting in a Signal Lower than Expected 28 APPENDIX I SOLUTIONS FOR NUCLEIC ACID BLOTTING amp HYBRIDISATION PROCEDURES eese 29 APPENDIX Il FACTORS AFFECTING STRINGENCY OF HYBRIDISATION 56 06110115 Pee 32 Effect of Temperature amp Salt Concentration 33 Summary of Conditions Affecting Hybridisation 33 APPENDIX III HYBRIDISATION BOTTLE 5 2 22 222 35 35 Ge 35 APPENDIX IV REFERENCES 36 HYBRIDISATION BOTTLE SAFETY It is important to follow correct safety procedures when using Hybridisation Bottles Please see Appendix l for details Thermo Scientific May 2003 Issue 7 CHAPTER 1 HYBRIDISATION GUIDE INTRODUCTION The immobilisation of nucleic acids on a solid support and subsequent detection of specific sequences of interest is one of the fundamental techniques of modern molecular
64. nutes per wash step 4 Repeat Steps 1 3 for each additional wash The wash solutions temperatures etc should be those recommended by the membrane manufacturer or refer to the Hybridisation Guide Note All wash solutions should be pre warmed for best results Washing in a Sandwich Some scientists prefer to remove the membranes from the bottles and wash Box them all in one container Washing may be performed very simply in the Shake n Stack on the shaking platform and is particularly useful when several membranes are being processed simultaneously 1 Remove the hybridisation bottle from the oven Pour off the hybridisation fluid 2 Using a pair of forceps carefully remove the membranes and place them in a suitable container with a lid e g sandwich box or similar 3 Using the forceps gently unroll the membranes 4 4 Shake n Stack Thermo Scientific Thermo Scientific Section 4 Methodology of Hybridisation Add a sufficient volume of the pre vvarmed first vvash solution to totally immerse the membranes Replace the lid and shake on the shaking platform in the Shake n Stack Oven at the required temperature for the specified time period Note Initial room temperature washes are not recommended and may result in subsequent background problems Remove the first wash solution and replace it with an equal volume of the prewarmed second wash solution Replace the lid and shake at the required temperature for the
65. nutes to fix DNA or UV cross link DNA Hybridisation Prehybridisation and Hybridisation are carried out in bottles in a Thermo rotisserie oven Better results have been obtained in bottles than in bags 1 2 10 Pre warm Hybridisation bottle containing 50ml 2 x SSC to 68 C Layer the following into a plastic box containing 50ml 2 x SSC a One piece of nylon mesh 23cm x 23cm Thermo Scientific o The membrane DNA side up o Two layers of nylon mesh d One piece of dummy Hybridisation membrane covering Hybridisation membrane below e One piece of nylon mesh This procedure prevents high backgrounds Mesh and dummy membrane are reusable after washing in distilled water Roll sandwich ensuring no air bubbles are trapped with DNA side facing inwards Place roll in Hybridisation bottle and carefully unroll sandwich again ensuring no air bubbles are trapped Tip off 2 x SSC and add 20ml prehybridisation buffer pre warmed to 68 C Prehybridise in rotisserie oven for 2 hours at 68 C Denature 300ng DIG labelled probe Add to 15ml prehybridisation buffer heated to 68 C Tip buffer off prehybridised membrane and add Hybridisation buffer to the bottle Hybridise overnight in rotisserie oven at 68 C Remove Hybridisation buffer and freeze This can be reused a further five times after heating to 95 C for 10 minutes Add 50ml 2 x SSC 0 1 SDS to bottle and roll in
66. opposite direction to release sandwich Thermo Scientific May 2003 Issue 7 21 11 Perform washes in bottle replacing each wash with in 50 100ml 4 x SSC 1 SDS at 68 C in 50 100ml 2 x SSC 0 1 SDS at 68 C 1 x 15 minutes in 50 100ml 0 1 x 0 1 SDS at 68 C SSC Alternatively remove membrane from roll and wash in plastic box 2 x 5 minutes in 500ml 2 x SSC 0 1 SDS at room temp in 500ml 0 2 x SSC 0 1 SDS at 68 C Thermo Scientific May 2003 Issue 7 22 CHAPTER 6 HYBRIDISATION GUIDE HYBRIDISATION USING OLIGONUCLEOTIDE PROBES The Hybridisation conditions outlined in Chapter 3 are generally applicable to probes derived by cloning or by gene amplification reactions ranging in size from 100bp to several hundred base pairs The Hybridisation conditions of synthetic oligonucleotide probes however are rather more problematical There are two types of oligonucleotide probes 1 A single oligonucleotide of a defined sequence derived from the nucleic acid sequence of the target gene A pool of oligonucleotides of degenerate sequences which are derived from the amino acid sequence of a region of the gene product of interest Due to the degeneracy of the genetic code i e most amino acids are specified by more than one triplet codon any particular sequence of amino acids will be specified by a number of different oligonucleotides Because the Tm of synthetic oligonucleotides is much lowe
67. r rating a a eher 250V7 LEROY as a 50 60Hz Fuse rating 110 120V 6 3A type T 220 240V 1 6A type T Ingress Protection Rating IP22 Rotisserie speed control PCB fuse 500mA Note This item should not be serviced by the customer Working Environment 15 C to 25 C Caution Care should be taken when lifting the Oven It is advisable that help is sought when removing an Oven from its packaging and when stacking Ovens for use Caution During operation caution should be taken with moving parts that are accessible when the Oven door is open Thermo offers full service and technical support for all its products Alternatively contact your local authorised distributor Thermo Scientific Thermo Scientific Section 2 Unpacking and Installation Shake n Stack Oven 4 Leveling Feet Shaking Tray Models 6240 6241 6245 Shaking Tray Drive Arm Assemb y Models 6240 6241 6245 Shaking Tray Bracket Models 6240 6241 6245 Povver Cable Drip Tray Note If any item is missing or damaged contact Thermo or an authorised Thermo distributor Warning If it is intended to use the Oven with radioactive isotopes the oven must be located in a designated Radiation Area Radiation safety procedures must be followed at all times n the event of spillage refer to Section 6 for guidance on cleaning and decontamination All Hybridisation Oven standard rotisseri
68. r than for longer probes the stringency of Hybridisation and washing procedures must be reduced and adjusted according to the base composition of the probe An approximate value for the Tm of an oligonucleotide probe has been derived by Wallace et a 1979 Tm C 2 x number of AT base pairs 4 x numbers of GC base pairs Hybridisations are carried out at 5 C below this temperature For a degenerate pool of oligonucleotides Hybridisation is carried out at 5 C below the lowest Tm for the set of sequences Prehybridise the membrane in oligonucleotide prehybridisation buffer 6 x SSPE SSC 10 x Denhardt s reagent 50ug ml denatured salmon sperm DNA at the Hybridisation temperature for at least one hour Add the radio labelled probe solution to the preHybridisation buffer Alternatively remove a small amount of preHybridisation buffer add the probe to this and replace this solution in the Hybridisation vessel Some researchers may prefer to use fresh Hybridisation buffer Hybridise with agitation or by rotating in bottles for approximately 12 hours at the Hybridisation temperature Thermo Scientific May 2003 Issue 7 23 4 Stringency washing is carried out as follows using large volumes at least 50ml of the following solutions pre warmed to the required temperature 2 x 15 minutes with 6 x SSPE 0 1 SDS at the Hybridisation SSC temp 1 x 2 minutes with 6 x SSPE 0 1 SDS at the Tm SSC 5 Wrap the mem
69. rmamide with a 5096 GC rich 500bp probe the Tm is calculated to be 101 C i e Hybridisation temperature 75 C Substituting the 6 x SSC with 1 x SSX in this system i e during washing reduces the Tm by 13 C Including 5096 formamide in each solution would have the effect of reducing each Tm by 30 5 C A shorter probe length 50bp reduces the Tm by a further 10 C In practice it is not necessary to calculate the Tm accurately for every system and the standard conditions given in the protocol sections will give good results However the equation does give a useful indication of the effect of adjusting the different variables on the stringency In general hybridise at low stringency then during washing increase the stringency by keeping the temperature constant but decreasing the salt concentration with each wash step Thermo Scientific May 2003 Issue 7 32 Effect of Temperature amp Salt Concentration HIGH STRINGENCY LOW Temperature Monovalentation concentration TO Temperature Monovalentation Summary of Conditions Affecting Hybridisation Temperature The Hybridisation temperature is one of the most critical factors because it affects the rate of hydrogen bond formation between DNA strands the hydrogen bonds in effect overcome the repulsive forces between the negatively charged strands If the temperature is increased then the stringency of Hybridisation is increased lonic Strength Increasing the monovalent
70. rotect the bottle and sealing area Replace O rings when worn or leaky Replace all O rings every six months Wear protective gloves to protect your hands in the event of accidental breakage Never over tighten caps on bottles Hand tight only is sufficient If the bottle 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 E ANANAAN RI The bottles should not be used at temperatures above 70 C NB Extra large bottles 70mm diameter are supplied with a vented cap to prevent excessive pressure build up during use A 0 45um hydrophobic tissue culture filter should be fitted to the vent outside the bottle as an added safety measure Thermo Scientific May 2003 Issue 7 35 APPENDIX IV REFERENCES Church G M and Gilbert VV 1984 Genomic sequencing Proc Natl Acad Sci 81 1991 Khandjian E W 1987 Optimised hybridisation of DNA blotted and fixed to nitrocellulose and hybridisation membranes Biotechnology 5 165 Grunstein M and Hogness D S 1975 Colony hybridisation A method for the isolation of cloned DNAs which contain a specific gene Proc Natl Acad Sci 72 3961 Benton W D and David R W 1977 screening gt recombinant clones by hybridisation to single plaques in situ Science 196 180 Southern E M 1975 Detection of specific sequences among DNA fragments separated by gel electrophores
71. rse the membranes Replace the lid and shake on the shaking platform in the Hybridisation Oven at the required temperature for the specified time period NOTE Initial room temperature washes are not recommended and may result in subsequent background problems 5 Remove the first wash solution and replace it with an equal volume of the pre warmed second wash solution Replace the lid and shake at the required temperature for the required time period 6 Step 5 should be repeated for any subsequent wash The washing protocol using the shaking platform will be identical to that used in the bottles Please refer to Chapter 7 for additional information on Washing Thermo Scientific May 2003 Issue 7 CHAPTER 4 HYBRIDISATION GUIDE NUCLEIC ACID PROBES There are now several types of nucleic acid probe available to researchers and a wide variety of radio labelling techniques for labelling these probes For convenience the types of nucleic acid probe may be classified as follows 1 Double stranded DNA probes Single stranded DNA probes RNA probes Synthetic oligonucleotides Double Stranded DNA Probes Any suitable DNA molecules cloned or uncloned can be used as a Hybridisation probe for example insert fragments from a DNA library may be excised from plasmids or bacteriophages by restriction enzyme digest and then labelled for Hybridisation Another possibility is to use the Polymerase Chain Reaction to synthesize copies of th
72. s 1490101 aum awes v 1e 086 ou 06 AjueueM au os aun Bulddiys 10 swoj e SIU Aug ano wo peddius s 3ueuudinba ino opp ay W014 989 OM SHEJS ponad AjuelleAA au ALNVddVM LONGOdd GUVGNVLS 1111183105 d3HSI3 0 83111 Thermo Scientific Shake n Stack A 6 60 7 3 L006 091 uoneoijdde 610945 pue eoueuojureuJ uone Jedo Ajue amp Jew 1ueuudinbe uo suonsenb noA 2 O peai L AA uoneuuoJur AUS UEAA 10 Jo1nquisip e2o 398 UO9 eoueuesjureu 9 1708 870 pue uoneJedo uonejejsui jueuudinbe ile p Ajjnjaseo sjenueuu uononjjsui Dall seAuJe jueuudinbe jno uoneuuojur uonejedoeJud ous eArisueueJdujoo uM djay oj Apes si sejes e90 INOA SsjonpoJd Jo sso Jo sjiyoad so o jnouwM seDeuiep jenuenbesuoo Jo 1998Jipui Aue 10J e qei eq jou eus oway AlddV TIVHS 38044 4 YVINDILYVd V G Z SSANLIS ALNIGVLNVHOYAW AO SAILNVYYVM ON Qaridiwi MO 1 N3lLINM YSHLSHM SSILNVYYVM 177 3O NAN NI ANY 3 103 SI ALNVYYVM SIH L uoneunsep 803 peddius sped jueuieoe doaJ pue pied eDejsod ouueu o peuunjeJ eq snu sued Buiuuojuoo uou ile uondo s ouueu jw 1ueuidinbe o sjueuodujoo Aug Jo 104 8 Joud 8 6 3 jueuruede S LAH S 6014 00 eu po
73. s maintained between the sides of the oven and any other equipment or object The oven may be located in a fume cabinet or similar chamber provided the cabinet is not used to contain hazardous or volatile chemicals with low flash points Full details of Technical Specifications are given in Section 8 Note If the Shake n Stack Oven is not used as specified in this manual the protection provided by the equipment may be impaired Thermo Scientific Shake n Stack 2 3 Section 3 Set Up the Shaking Platform Drive Arm l Slot Assembly Sleeve Drive shaft g Screw Rotisserie drive shaft Drive arm Shaker peg Shaker peg Shaker tray Oven base Figure 1 Shaking Tray Components 1 The shaker bracket is installed at the factory If replacement should ever become necessary instructions follow Continue to Step 2 e Loosen existing screws at the bottom of the oven back sufficiently to allow slots in bracket to slide down onto screw shafts Retighten screws to secure back plate See Figure 2 Figure 2 Replace Shaker Bracket 2 Fit the rear right hand peg of tray into vertical slot at right of bracket allowing tray to temporarily rest on oven base Refer to Figure 3 Figure 3 Insert Tray Peg into Slot Thermo Scientific Shake n Stack 3 1 Section 3 Set Up the Shaking Platform 3 Slide drive arm assembly sleeve onto rotisserie drive shaft aligning location peg within drive arm sleeve
74. sfer solution Thermo Scientific May 2003 Issue 7 Northern Blotting Electrophoresis of RNA should be carried out in a denaturing gel system Maniatis et a 1989 gives details of running denaturing RNA gels using formaldehyde or glyoxal 1 After electrophoresis of the RNA in a denaturing gel the transfer can be set up as described for DNA gels see Southern Blotting above The denaturating step 3 and the neutralising step 4 of the gel are not required as the RNA is denatured in the gel However some researchers recommend reduced stringency denaturation and neutralisation steps to facilitate transfer of large RNA molecules as follows 50mM NaOH 10mM NaCl Gentle agitation of the gel is essential to prevent damage to the gel during these steps The shaker in the Shake n Stack Midi Dual 14 and Maxi 14 are ideal for this purpose 2 RNA transfer is carried out in 10 20 x SSPE using the same procedure as outlined for DNA gels in Southern Blotting section above 3 HAfter transfer of the RNA to the Hybridisation membrane is completed fixing of the RNA is carried out by baking at 80 C for 2 hours or by UV cross linking 4 After fixing the RNA the membrane is ready for Hybridisation Membranes not used immediately may be stored between sheets of Whatman 3MM paper in sealed plastic bags at 4 C Thermo Scientific May 2003 Issue 7 CHAPTER 3 HYBRIDISATION GUIDE HYBRIDISATION PROCEDURES Nucleic acid hybri
75. st 15 2010 15 Aug ist 20 ye Eric Pickrell Regulatory Compliance Specialist ThermoFisher SCIENTIFIC Rev 2 Background Reduction General Thermo Scientific Appendix Troubleshooting Guide for Nucleic Acid Hybridisations Our bottle system is intrinsically simpler and safer to use than other methods of hybridisation such as hybridisation in bags or plastic boxes In the Hybridisation Ovens the temperature of the solutions is precisely controlled and regulated and the rotisserie device ensures that the solutions are continuously and evenly distributed over the membrane Thus the optimum conditions for hybridisation and washing are maintained throughout when using this system However during the transfer of protocols from bags to bottles some minor adjustments to the protocols may be necessary When loading the mesh and membranes into the bottles air bubbles should be avoided Ensure that the oven is positioned on a flat surface so that the probe solution is distributed evenly along the length of the bottles and that there is sufficient probe solution to cover the entire membrane On occasion the mesh and membrane can become tightly rolled up in the bottle This occurs if the mesh is loaded incorrectly see Figure 4 1 in Section 4 All solutions for nucleic acid hybridisations should be prepared using distilled water and highest quality reagents in clean glassware In particular water with a h
76. t the membrane in 6 x SSPE SSC and place on dry filter paper Pipette samples into the wells for binding of the DNA to the membrane and follow manufacturer s instructions to operate apparatus correctly If samples are spotted manually apply 0 5 1 0ul aliquots and allow to dry between applications to prevent excessive spreading Dismantle the apparatus and remove the membrane Place the membrane on a sheet of Whatman 3MMVN filter paper saturated with denaturing solution for 1 minute Transfer the membrane to a sheet of filter paper saturated with neutralising solution for one minute Place the membrane on dry filter paper and allow to air dry Fix the DNA by baking the membrane for 1 hour at 80 C or by UV cross linking UV cross linking is the most efficient method of fixing the DNA to Hybridisation membranes and is particularly useful if reprobing is to be carried out After fixing the DNA the membrane is ready for Hybridisation Membranes not used immediately may be stored between sheets of Whatman 3MMrv in sealed plastic bags at 4 C Thermo Scientific May 2003 Issue 7 Colony Plaque Blotting Colony Blotting 1 Grow bacteria on agar containing the appropriate selective antibiotic until colonies are approximately 0 5 1mm in diameter Place a Hybridisation membrane cut to the size of the plate on to the surface of the agar Orientation points should be marked at this stage by piercing the membrane at three asymmetric poin
77. term reliability t is important to check your bottles regularly for chips stress fractures and cracks If these occur discard the bottle Ensure bottles are stored either in a suitable rack or with caps replaced between experiments This will protect the bottle and sealing area Replace O rings when worn or leaky Replace all O rings every six months e Wear protective gloves to protect your hands in the event of accidental breakage e Never over tighten caps on bottles Hand tight is sufficient Caution Dropping or over tightening lids can cause cracking and failure Lids should be inspected prior to each use e Ifthe bottle 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 e The bottles should not be used at temperatures above 70 C Shake n Stack A 5 t ed 60 7 L006 091 Uonpuloyul JO 7010017 SID 18201 1263002 ven v 1911 uoneaijdde 613905 pue 831 8 asueuazuiew uorjejedo AJUeeM 708001558 uo suonsanb mo Jamsue 0 Apea L M 9 5 0p 10 epeue pue ysn 287 86 008 1e 14901 80 99 e31uu29 ile asea d pauinbai s ao n as 1uauidinbo J QOUBUB UIEW 8 708 80 pue uone1edo uoiejjezsui queuudinba jreyep Ajjnjaseo sjen UPW uonnujsur peuug seAuJe 1ueuudinbo jno 210 9q uoneuuojur 0 76260820
78. tion 73 Leen eng Figure 5 1 Variable Axis Rotation Shake n Stack 5 1 Section 5 Rotisseries 50ml amp 15ml Tube Rotisseries are available that can hold 0 5 fl oz 15ml and or 1 7 fl oz Rotisseries 50ml disposable tubes Details are given in Section 8 Tubes should be inserted into the rotisseries by sliding the tube sideways into the rotisserie Care should be taken not to push the tubes too firmly into the rotisseries otherwise the tube will crack 5 2 Shake n Stack Thermo Scientific Section 6 Cleaning and Decontam nat on All items which come into contact with a labeled probe should be decontaminated before re use In many cases a short rinse in water will be sufficient In some instances however further decontamination procedures will be required Meshes The quantity of radioactivity adhering to the mesh can be minimised by leaving the mesh in with the membrane throughout the washing procedure Any radioactivity remaining can then be stripped from the mesh by the following procedure 1 Strip wash the mesh by incubating it in distilled water at 65 C in a shaking water bath for 15 minutes Repeat 2 Ifthe mesh is still contaminated soak it in a diluted solution such as Decon 90 overnight 3 Remove the detergent and proceed with two washes in distilled water for 10 minutes each 4 If the mesh has been decontaminated no further action is necessary If however the mesh is still contami
79. tisserie action uniformity within the bottle is 0 25 C However it is important to note that the temperature inside the oven chamber will vary from point to point and therefore the temperature of objects vessels which are not placed in the rotisserie will vary from that which is displayed Refer to Setting the Required Temperature for recommendations on temperature setting when using shaking platforms Shake n Stack 1 1 Section 1 ntroduction 1 2 Oven Temperature Monitoring cont d Safety Precautions Shake n Stack The Shake n Stack is designed to be used with Thermo s hybridisation bottle These high quality leak proof containers can be used at temperatures up to 70 C for catalogue numbers see Section 8 Bottles should not be used above 70 C without taking the precaution of releasing the pressure formed by the release of gases from solutions as they are warmed Simply loosening then retightening the bottle cap at approximately 60 C should relieve the pressure Caution Dropping or over tightening lids can cause cracking and failure Lids should be inspected prior to each use Caution Always wear gloves when handling the bottle and use the bottle gripper provided see Appendix II for full instructions on Bottle Care Finally the Shake n Stack Oven is designed for reliability and ease of maintenance The rotisserie shaking platform and drip tray can be easily removed for cleaning Supply Powe
80. to a hybridisation bottle and then insert the roll in such a way that the leading edge inside the roll and the trailing edge are positioned relative to each other as shown in Figure 4 3 Place the bottle on a flat surface then slowly unwind the membrane and mesh around the inside of the bottle by gently rocking and rolling the bottle along the surface No air bubbles should be visible between the membrane and the bottle If bubbles are present the membrane should be removed and re rolled The procedure should then be repeated more gently Rock the bottle backwards and forwards to attach the first part of the membrane to the bottle Then roll the bottle to unwind the mesh and membrane Continue until the membrane and mesh are fully unwound The mesh ensures the probe will have access to all parts of the hybridisation membrane including those parts of the membrane which overlap Shake n Stack 4 1 Section 4 Methodology of Hybridisation Insert Membranes in a Assemble these items Bottle e Shallovv tray containing 2 x SSPE SSC e Mesh Membrane Roll up mesh and membrane Figure 4 1 2 Place 0 3 0 5 fl oz 10 15ml of SSPE SSC into a bottle and then insert the roll centrally Figure 4 2 Zei Figure 1 Roll Mesh and Membrane Figure 4 2 Insert Roll 3 Secure cap and holding bottle horizontally roll to catch the trailing edge of the mesh continue rolling in the same direction until coil of mesh and me
81. ts with a sterile needle Replica filters may be prepared from the initial colony lift at this stage by pressing a second Hybridisation membrane firmly on to the template membrane for a few seconds The simplest way to do this is by sandwiching the membranes between glass plates lined with a few sheets of Whatman 3MM paper and pressing them firmly together Carefully separate the membranes to avoid smearing the colonies This replica and any subsequent replicas should also be marked with orientation points The replica membranes are then placed colony side up on to fresh agar plates containing the appropriate selective antibiotic and incubated at 37 C until colonies of 0 5 1mm are present on the surface of the membrane At this stage it is possible to amplify low copy number plasmids relative to cellular DNA by transferring the filters on to agar plates containing chloramphenicol at 200ug ml and incubating for 10 hours at 37 C Place the Hybridisation membranes on Whatman 3MM paper saturated in neutralising solution for 3 minutes then repeat with fresh neutralising solution Rinse the membranes briefly in 2 x SSPE SSC then air dry on a sheet of filter paper Fix the DNA to the membranes either by baking for two hours at 80 C or by UV crosslinking After fixing the DNA membranes may be stored between sheets of Whatman 3MM paper in sealed plastic bags at 4 C Prior to Hybridisation it may be advantageous to carry out a pre w

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