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1. H s Thermo Scientific Ordering Information Thermo Scientific Section 8 Specifications 110V Model 6246 220V Model 6247 Includes a 1 4 in 35mm stainless steel rotisserie drip tray 4 medium bottles 1 pack mesh 1 large bottle gripper orbital platform and linear platform 220V Model 6249 Includes a 2 8 in 70mm stainless steel rotisserie drip tray 4 extra long bottles 1 pack mesh 1 extra long bottle gripper orbital platform and linear platform m r IE 1 7 fl oz 30 x 50ml tubes and 0 8 x 0 5 fl oz 25 x 15ml tubes XE 0 54 x 1 7 fl oz 16 x 50ml tubes and 1 7 x 0 5 fl oz 50 x 15ml tubes PA CECI SE Hybridisation Oven 8 3 Section 8 Section title 8 4 Hybridisation Oven Additional Accessories continued Hybridisation Bottles Extra Long Bottle 2 8 x 11 8 in 70 x 300mm Long Bottle 1 4 x 11 8 in 35 x 300mm Medium Bottle 1 4 x 9 8 in 35 x 250mm Short bottle 1 4 x 5 9 in 35 x 150mm Bottle Gripper 11 8 in 300mm for extra long bottles Bottle Gripper 11 8 in 300mm for long medium bottles Bottle Gripper 5 9 in 150mm for short bottles Nylon Mesh 5 Large mesh sheets 9 x 9 in 23 x 23cm 5 Small mesh sheets 4 x 6 in 10 x 15cm Conforms to IEC1010 Pollution Degree 2 110094 110095 110096 110097 222055 2200056 2200057 222053 222052 Conforms to Electrical Safety Standards described in IEC1010
2. i 18 RNAGPIODES Lia ali 19 Synthetic Oligonucleotides i 19 NON RADIOACTIVE SYSTEM eene 20 The DIG System in Thermo Hybridisation Ovens 20 Electrophoresis 8 Transier eee 21 DNA Hybridisation casona cede a dates 21 HYBRIDISATION USING OLIGONUCLEOTIDE PROBES 23 Thermo Scientific 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 iac sued 26 Probe Preparatorias 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 PEACION LA Pee 32 Effect of Temperature 8 Salt Concentration 33 Summary of Conditions Affecting Hybridisation 33 APPENDIX III HYBRIDISATION BOTTLE SAFETY 222 35 Use of the Bottle Gripper clic 35 i 35 APPENDIX IV REFERENCES 36 T HYBRIDISATION BOTTLE SAFETY It is importa
3. Thermo Scientific O O O DX Qj w N 0 MU w N 20 MODELS 6246 amp 6248 110 VAC 50 60Hz IPH 2 3FLA of 7 9 e e FRA 900959 s l 1 x 7 2 USL E m ali 1 CR 2 L b Ui lo 16 CONTROL RELAY 300374 257 T STAT 100 400172 FERRITE 4 PLACES 270164 360259 12 23 RECIPROCATING SWITCH CAPACITOR luF 170044 et H 3 1 RECIPRO MOTOR CI CATING ONTROL Q 34 33 32 ROTISSERIE SWITCH 360259 22 4 CAPACITOR luF 2 fo 36 1 ROTISSERIE SPEED CONTROL 38 170044 a LINE CORO 430108 LINE FILTER FUSE HOLDER 460186 FUSES 6 34 TYPE T 2 REQ D 230210 POWER SWITCH ROCKER DPST 360258 BRANCH POWER T STRIP 1 370599 TOROIDAL TRANSFORMER 115V IN 230V DUT 420114 INTERIDR CHAMBER FAN 156130 CHAMBER HEATER 250WATT 240V 137023 OVERTEMP RELAY 300375 16 TEMP SENSOR 100 DHM PLAT 290186 TEMP CONTROL 231216 BRANCH POWER T STRIP 2 370599 RESISTOR 100 OHM 1210101 RECIPROCATING MOTOR 24VDC 156132 RECIPROCATING
4. Thermo Scientific Setting the Required Temperature cont Thermo Scientific Section 2 Unpacking and Installation The Maxi 14 Hybridisation 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 Chapter 8 Note If the Maxi 14 Hybridisation Oven is not used as specified in this manual the protection provided by the equipment may be impaired A Hybridisation Oven 2 3 Placing Membranes Thermo Scientific in a Bottle Section 3 Methodology The following paragraphs detail the methodology of hybridisation using the Maxi 14 Hybridisation Oven Place the bottles to be used in the Oven during the vvarm up period approximately 1 hour 2 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 Chapter 8 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 0 8 x 0 8 in 20 x 20mm mem
5. 8 75MM BROW 33 5MM GREY 9 Du BLUE 34 5MM PED 0 5MM BROW 35 5MM RED 1 75M GRN YEL 36 5MM BLACK 2 1 75MM BROW 37 BMM PINK 3 75MM BLUE 38 SMM PINK 4 75MM BLUE 39 75MM BLUE 5 5MM BROW 40 75MM BROWN 6 5MM BROW 41 75MM BLUE 7 1 75MM BLUE 42 5 GRN YEL 8 75MM BROW 9 22MM RED 20 22MM BLUE 21 75MM BROW 22 75MM BROW 23 75MM BLUE 24 75MM BROW 25 75 BROW ROTISSERIE SELECT RESISTOR VALLES CHART SYM OHMS STK NUMBER RI 511 205110 R2 680 21068 R3 820 21082 R4 K 210102 R5 1 2K 210122 R6 1 20130 R7 1 6K 20160 R8 1 8K 20180 Rg X 20200 RIO 2 2K 20220 RII 2 4K 20240 Electrical Schenatic cal mes e gt Model S 6246 6247 su n cereum a aee oe ree ee as Mt TES ER ony oo fw eue ebe sme MOK 1 Der ELECTROSTATIC ThermoFisher cz 6247 70 0 D REV 2 SENSITIVE DEVICES SCIENTIFIC TOLERANCE UNLESS OTHERWISE SPECIFIED DRAWING NUMBER SIZE D 5 BOX 649 MARIETTA DHID 45750 ANGLES DECIMAL ct 6247 70 0 D age 3 of 3 Thermo Scientific Hybridisation Oven 9 3 Troubleshooting Guide for Nucleic Acid Hybridisations Thermo Scientific Section 10 Appendix 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 Maxi 14 Hyb
6. 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 the 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 Kd 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 electro
7. KE ti d ete ay na Pa va d 3 4 Method 1 Washing in Hybridisation Bottles 3 4 Method 2 Washing in a Sandwich Box 3 4 Other Procedure aeneae suo see pat du Grote nd ars a ete aco 3 5 Shaking Incubations 4 1 Rotisserles XY iii annie veces A ia a R Y 5 1 1 7 amp 0 5 fl oz Tube Ro sseries sai er aia 5 1 Cleaning and Decontamination 6 1 Bottlesang Gaby vec r ok dvd elia ire tar io iI 6 1 Mes AA Ga edn s he coat us Rose de ai 6 1 Kot ee e NN iR WP weed E VEN es E 6 2 Oven Interior Shaking Platform cao s s hei 6 2 Use of Radioactive Probes 7 1 Specifications cgn sp e Ka Ra 0 herent Na 0 aa R RF XR ERE 8 1 Ordering Information ode AS Rete o ie 8 3 Electrical Schematics 9 1 Appendix ici cower sare RR Eege EH 10 1 Warranty eec 11 1 Hybridisation Oven V 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 including detailed protocols is contained in the Hybridisation Guide Hybridising in bottles means that probe volumes may be significantly reduced compared to expe
8. 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 washing step to remove colony debris and hence reduce background noise Thermo Scientific May 2003 Issue 7 Pre washing is carried out as required by incubating in at least 200m of prewash solution see Appendix 1 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 p
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10. probe degraded This is more likely to occur when using RNA probes 7 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 8 Hybridisation and or washing conditions were too stringent i Increase salt concentration ii Decrease temperature iii Reduce concentration of SDS iv Reduce wash times 9 The hybridisation time was too short Thermo Scientific Hybridisation Bottle Safety Thermo Scientific Section 10 Appendix Il Use of the Bottle Gripper Part No 222057 for 6 in 15cm short bottles Part No 222056 for 11 8 in 30cm medium or long bottles Part No 222055 for 11 8 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 Helping you to safely tighten and unscrew caps e Making handling of hot bottles easier For more details and ordering information contact the Technical Services Department or your local Thermo Distributor Bottle Care Our Hybridisation Bottles are made of thick walled borosilicate glass which protects users from radiation and has excellent long term reliability tis important to check your bottles regularly for chips stress fractures and cracks If any of these are noted discard the bottle Ensure bottles are stored either in a suitable rack or with
11. 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 lower 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 temper
12. 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 points 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
13. and replaced with 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 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 t
14. 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 with 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 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 will determine if the final wash should be carried out 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 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
15. any damages direct or incidental arising out of or related to the use of this manual 2003 Thermo Scientific All rights reserved Hybridisation Oven Thermo Scientific Preface Important operating and or maintenance instructions Read the accompanying text carefully Potential electrical hazards Only qualified persons should perform procedures associated with 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 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 Thermos compliance with this directive the recyclers in your country and information on Thermo products will be available at www thermofisher com Y Always use the proper protective equipment clothing gloves goggles etc VY Always dissipate
16. 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 Wear protective gloves to protect your hands in the event of accidental breakage 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 A Caution 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 A Caution The bottles should not be used at temperatures above 70 C A Hybridisation Oven 10 5 Declaration uf Contormity Manufacturer s Name Thermo Fisher Scientific Manufacturer s Address 401 Millcreek Road Marietta Ohio 45750 U S A Product Description Maxi 14 Hybridization Ovens Product Designations 6246 through 6249 Year of Initial Marking CE 2005 Affected Serial Numbers Release 1 Release Level REL shown on Serial Tag These products conform to the following European Union Directive s EMC 89 336 EEC LVD 73 23 EEC These products conform to the following Harmonized International and National Standards EMC LVD EN 50081 1 1992 EN 60335 1 1994 EN 50082 1 1992 Richard L Miller CQE Regulatory Compliance Manager ThermoFisher SCIENTIFIC 27 February 2007 Rev 1 Section 11 Warranty
17. 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 Kinase 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 fragme
18. repeated for any subsequent wash The washing protocol is obviously the same as is currently being used 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 l 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 NaCl 0 5M NaOH 30 minutes iii Neutralisation 1 5M NaCl 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 hence 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 Probe Stripping from Filters after Hybridisation An example of probe stripping procedure is as follows Wash the filter at 65 C in 5mM Tris Cl pH8 0 0 2mM EDTA 0 1 x Denhardt s reagent for 1 2 hours Check for residual activity by autoradiography then re probe Hybridi
19. 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 electrophoresis 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 clonin
20. without a service call When more extensive service is necessary we will assist you with 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 Scientific Controlled Environment Equipment 401 Millcreek Road Box 649 Marietta OH 45750 International customers please contact your local Thermo Scientific distributor iv Hybridisation Oven Thermo Scientific Section 1 Section 2 Section 3 Section 4 Section 5 Section 6 Section 7 Section 8 Section 9 Section 10 Section 11 Thermo Scientific Table of Contents Introduction ioni Ici bu ERREUR ETT 1 1 Oven Temperature Monitoring ses charts ase oA a POT APA SUR S 1 1 Unpacking and Installation 2 1 Setting the Required Temperature d Ze dii 2 2 JDGI3UDI o5 oed AS vado Neck tru ei dau 2 2 Methodology ea tex einer caw na eei md 3 1 Placing Membranes in a Bottle us ara tare edet Ac 3 1 RO RARO SOR A He A RE 3 2 Pre Hybridisatiom EE EE 3 3 FHiybridisation atc da quu urat Pet rata t 3 3
21. 2003 Issue 7 28 APPENDIX 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 H20 27 69 7 49 Dissolve in 800ml of H20 and adjust pH to 7 4 with NaOH solution Adjust the volume to 1 litre with H20 and sterilise by autoclaving 3 100 x Denhardt s Reagent Ficoll 29 Polyvinyl pyrollidone 2g Bovine serum albumin Fraction V 2g Dissolve in 100ml H20 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 NasEDTA Prehybridisation Buffer for Southern Blots Prepa
22. Department or an authorised Thermo distributor Warning If it is intended to use the Oven with radioactive isotopes it must be located in a designated Radiation Area Radiation safety procedures must be followed at all times In the event of a spillage refer to Chapter 6 for guidance on cleaning or decontamination Maxi 14 Hybridisation Oven standard rotisseries are designed to accept our long medium or short hybridisation bottles extra rotisseries are available to accept extra long 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 Connect the Oven to the mains power supply using only the removable mains line cord supplied The line cord should be fitted with a 10A fuse for both 110 120V and 220 240V applications The Oven is grounded via the mains line cord and should only be connected to an grounded supply See Specifications Thermo Scientific Hybridisation Oven 2 1 Section 2 Unpacking and Installation Operation Setting the Required Temperature 2 2 Hybridisation Oven The three switches found on the control panel operate the power rotisserie and shaking platform 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 a green section of the switch is exposed to indicate that the Oven is ON The swi
23. Information 0343191939 20 2 d L006 OSI uoneuuojJul AjueJJeM 10 JOJNQUISIP 890 JOe UOD ysn 24 apisino uoneoidde jeloeds pue 2 soueuajuiew uoneJedo AjueJem jueuudinbe uo suonsanb noA Jamsue 0 peaJ 9J 9M 9 5 0v L Jo epeuey pue VSN 064 1 12 888 1 1e juauyiedag sanag eoiuuo9 peo aseajd p nb s sones 1ueujudinbo y QOUBUd UIEW eAnueAeJd pue uoneJjedo uonelleysul juawdinba rejep AjjinjaJeo sjenuew uononujsul Doll seAuJe jueuudinbe unoA aJojeq uoneuuojui uoneJedaJd aus aaissuayalduos yum dieu O pea si au Sales ouuoeu 197201 INOA sjonpold JO sso Jo SHJOIA 1901 o uoneyu moyyum Buipnjui sobewep jenuenbesuoo Jo 0 1 Aue 10 e jqei eq jou jeus oway AlddV TIVHS 39044 4 YVINDILAVA V Od SSINLIA YO ALNIGVLNVHOYAW JO SAILNVYYVM ON 031141 YO 1V3O N3LLINM Y3HLIHM 95 Y3HLO 177 JO NANT NI ANY 3AISN19X3 SI ALNVYYVM SIHL uoneunsep gO peddius ale sued juawsaoe dal pue pied aBejsod uonejodjo2 uodJoe 3 oway peuunjaJ eq jsnu sued Buiuuojuoo uou ile uondo s ouueu yy 3ueuudinbe Jo sjueuodujoo Aug jo JO JeAoudde Joud auf snw juswWyedeq 590 eoiuuo9 eu poled AjueJem euiBiuo ay puo q ped juauoduo9 y Jo jueuudinbe y Jeyue O AjueJew y DU X JOU Jeus jueJem sig Jepun juawidinba JO sued sjueuodujJoo jo ed JO jueuieoejdes AUE HEA SIU WO
24. MOTOR CONTROL 191779 RECIPROCATING SPEED CONTROL 4 7K 310189 RESISTOR 100 OHM 1210101 ROTISSERIE MOTOR 24VDC 156128 ROTISSERIE SPEED CONTROL 191778 ROTISSERIE SPEED SELECT SWITCH 360262 RI RII SEE RESISTOR VALUES CHART Electri cal Schenatic Model 6246 6247 6248 6249 Moxi 14 Oven 6247 70 0 D REV 2 Pag e of 3 Thermo Scientific Hybridisation Oven 9 1 MODELS 6247 amp 6249 220 VAC 50 60Hz 1PH 1 1FLA 39 LINE CORD COUNTRY DEPENDENT 40 0 41 l ws 2 LINE FILTER FUSE HOLDER 40 MY di 480186 42 FUSES 1 BA TYPE T 2 42 230206 i POWER SWITCH ROCKER DPST 43 3 H 360258 44 42 E 45 lt H7 46 fi 47 x 8 Seu 68 BRANCH POWER T STRIP 1 48 P IRR 370599 q fu HLE TL 30 5 NU INTERIOR CHAMBER FAN L 2 156130 52 o E A E CHAMBER HEATER 250WATT 240V owe 38 137023 as 53 o CONTROL RELAY SAFETY T STAT 100C 300374 400172 54 19 CR I DVERTEMP RELAY 20 300375 51 55 TEMP SENSOR 100 OHM PLAT FERRITE 4 PLACES 290186 56 2 26 270164 Lo 28 28 57 1 27 9 6 6 0 9 o 5
25. Maxi 14 Hybridisation Oven Operating and Maintenance Manual 7006246 Rev 4 Visit us online to register your warranty Thermo www thermoscientific com warranty SCIENTIFIC Preface Models Covered Model Voltage Rotisserie 6246 110V 1 4 inch 35mm 6247 220V 1 4 inch 35mm 6248 110V 2 8 inch 70mm 6249 220V 2 8 inch 70mm MANUAL NUMBER 7006246 4 25799 0V 325 10 28 09 Added caution not to over tighten lid of bottle CCS 3 24622 0V 292 5 15 08 updated elecschematics to show country dependent line cord CCS 2 23459 6 12 06 Updated bottle to 3 2 wall thickmness added X ray warning CCS 1 12 2 03 Added Model 6248 6249 new rotisserie 70mm CCS 0 Original CCS REV ECR ECN DATE DESCRIPTION By Thermo Scientific Hybridisation Oven 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 A Caution All internal adjustments and maintenance must be performed by qualified service personnel A Material in this manual is for information purposes only The contents and the product it describes are subject to change without notice Thermo Scientific makes no representations or warranties with respect to this manual In no event shall Thermo be held liable for
26. S 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 Hybridisation Oven 10 3 Section 10 Appendix Summary of Factors Resulting in Lower Signal Than Expected 10 4 Hybridisation Oven Summary of Factors Resulting in a Signal Lower than Expected in Insufficient exposure time of membrane to film during autoradiography 2 Inefficient transfer and or binding of nucleic acids to the nylon membrane 3 Target sequence present at very low copy numbers Increase the amount of sample loaded on to the gel 4 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 5 No probe homology 6 Double stranded DNA probe was not denatured see standard protocols Alternatively
27. ap s juaudinba noA aun aues ay A ojeuurxoJdde ye 10 out IV jueJem ay os euin Buiddiys 10 smojje siu AyIoe uno Wo peddius si juawdinba ino ajep ay WO syjuou OM suejs pollag AueJeM SUL ALNVSHSHVM H31V3G IWNOILVNYALNI OISILNAIDS 43HSIA ONYIHL Thermo Scientific Hybridisation Oven 11 2 HYBRIDISATION GUIDE USER INSTRUCTION MANUAL Manual 7222060 Rev 0 CHAPTER 1 CHAPTER 2 CHAPTER 3 CHAPTER 4 CHAPTER 5 CHAPTER 6 HYBRIDISATION GUIDE Contents INTRODUCTION 1 NUCLEIC ACID TRANSFER PROCEDURES 3 BorsloetBletgs a serale aria 3 Colony Plaque Blotting AEN 4 Plaque Biotihdee a A 5 lef E Bee l DE 5 Capillary Elotiing een EE 6 Figure 2 1 Capillary Blot Assembiy 7 Northern BIO RING assi 8 HYBRIDISATION 5 2 22 9 Southern Blot DNA HybridisatioNS eee 9 Northern Blot RNA HybridisationN 11 Notes for Nucleic Acid Hybridisations using the Thermo Range of EQUIPMENT iii aabt 12 Placing Membranes in a Bopien 12 Figure 3 Inserting Membranes in a Bottle 14 PrehyDridIsalOE een a Aaaa daaa 15 FAV ORIGIS QUO Mes e tect reds aa AAA 15 NEC E 16 NUCLEIC ACID PROBES c nica 18 Double Stranded DNA Probes i 18 Single Stranded DNA Probes
28. ashes 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 stage 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 1 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
29. ature 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 2x15minutes with 6 x SSPE 0 196 SDS at the Hybridisation SSC temp 1x2minutes with 6 x SSPE 0 196 SDS at the Tm SSC 5 Wrap the membrane 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 Q 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 proto
30. branes can be hybridised in a single hybridisation bottle 5 Place 0 34 0 51 fl oz 10 15ml SSPE SSC into a hybridisation bottle 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 3 6 Place the bottle on a flat surface 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 backward and forwards to attach the first part of the membrane to the bottle Then roll the bottle in order to unwind the mesh and membrane 7 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 Hybridisation Oven Section 3 Methodology Insertion 1 Needed Shallow tray containing 2 x SSPE SSC mesh membrane 2 Roll up mesh amp membrane 3 Place 10 15mls of SSPE SSC into a bottle then insert the roll centrally 4 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 membrane is well positioned 5 Pour ou
31. 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 Hybridisation Oven 10 1 Section 10 Appendix 10 2 Hybridisation Oven Washing Procedure Stringency washing should be carried out as follows using large volumes approximately 3 4 fl oz 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 Di 1 x 30 minutes with 2 x SSPE SSC 0 1 SDS at 65 C 3 1 x 10 minutes with 0 1 x SSPE SSC 0 1 SDS at 65 C AII wash solutions should be pre warmed to the appropriate temperature An initial room temperature wash is not recommended and this 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 ra
32. cols 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 the 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 Washing Procedure Stringency washing should be carried out as follows using large volumes approximately 100ml of the fol
33. d then chilling on ice Add the denatured salmon sperm DNA to the buffer to a final concentration of 50ug ml 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 membrane 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 0 196 505 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 w
34. e 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 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 rea
35. e 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 Summary 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
36. extreme cold or heat and wear protective clothing Y Always follow good hygiene practices Y Each individual is responsible for his or her own safety Thermo Scientific Hybridisation Oven iii Preface Do You Need Information 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 1 740 373 4189 FAX http www thermo 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
37. g 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 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 Itakura K 1979 Hybridisation of synthetic oligodeoxyribonucleotides to 174 DNA the effect of single base pair mismatch Nucleic Acids Res 6 3543 Wood W 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 Scientific Controlled Environment Equipment 401 Millcreek Road Marietta Ohio 45750 United States www thermofisher com
38. gent 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 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 111 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 borosilica
39. he 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 Washing 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 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 fr
40. ing platform can be decontaminated by wiping clean with Decon 90 followed by distilled water rinse 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 6 2 Hybridisation Oven Thermo Scientific Section7 Use of Radioactive Probes The Maxi 14 Hybridisation Oven has been tested with a variety of non radioactive probes e g the DIG Non Radioactive Nucleic Acid labelling and detection system from Boehringer Mannheim Refer to the Hybridisation Guide for guidelines on the use of non radioactive systems Warning Refer to notes on the use of hazardous chemicals in the Warranty section A Thermo Scientific Hybridisation Oven 74 Section8 Specifications Display LED Accuracy at 55 C 1 0 Safety Overtemp Cut out Rotisserie Material Stainless steel shaft variable axis 0 15 stainless steel rotisserie wheels Variable speed rotisserie Yes Rotisserie speed 5 15 RPM Shaking Platform Yes 7 Eso a xa nternal 13 8 VV x 20 1 H x 12 6 D 350mmW x 510mmH x 320mmD Thermo Scientific Hybridisation Oven 8 1 Section 8 Specifications 8 2 Hybridisation Oven Tea lm CN 1 ma 1 reen sa Hisse i Internal 13 8 W x 20 1 H x 12 6 D 350mmW x 510mmH x 320mmD frese lim
41. 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 hybridisations 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 an
42. lowing 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 1x30 minutes with 2 x SSPE SSC 0 1 SDS at 65 C 3 1x10 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 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 10 cpm 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 Thermo Recovery kits are excellent for thi
43. ly 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 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 following 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 t
44. m MMC TEMP CONTROL fos ee eee SH 231216 58 TTD Re LUN 59 l 24 MEE 60 E A Li BRANCH POWER T STRIP 2 61 PP 370599 TL LT 62 E CAPACITOR luF RESISTOR 100 OHM 63 170044 1210101 c 30 RECIPROCATING MOTOR 24VDC 64 RECIPROCATING SWITCH KE 58 we 12 31 O 65 S 8 66 15 50 RECIPROCATING MOTOR CONTROL 191779 67 34 RECIPROCATING SPEED CONTROL 4 7K 310189 68 ES 69 70 CAPACITOR Jup RESISTOR 100 OHM 3 170044 1210101 ROTISSERIE SWITCH ROTISSERIE MOTOR 24VDC 72 156129 22 o k 73 La asse 1 Electrical Schenatic d LoS ROTISSERIE SPEED CONTROL 74 SPEED CONTROL de 8 191778 Model 8 ROTISSERIE SPEED SELECT SWITCH 6246 6247 E H g 7 75 lo SE 6248 6249 RI RII 76 2 To fs le em Je z s s SEE RESISTOR VALUES CHART Moxi 14 Oven 38 27 6247 70 0 0 REV 2 Page 2 of 3 9 2 Hybridisation Oven Thermo Scientific T 78 78 0 O LO cO WD wo l OOO 0 0 Uu N O K ul t C WIRE REFERENCE CHART NO SIZE COLOR NO SIZE COLOR 1 75MM BROW 26 75MM BROWN 2 75 BLU 27 75MM BLUE 3 75 BRO 28 SENSOR RED 4 75MM BLU 20 SENSOR WHITE 5 75MM BROW 30 5MV RED 6 75 BLUE 3l BMM BLACK 7 75MM BROW 32 5MM PINK
45. 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 minutes 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 pr
46. nbesuoo Jo joaJpul Aue 10 ejqei eq 104 IlEUS AlddV TIVHS 4SOdMund YVINDILYVd V SSINLIA YO ALNIGVLNVHOYAW JO SAILNVYYVM ON Qaridwi YO 1V3O NILLIVJM SSILNVYYVM YSHLO 177 1311 NI ANY 3AISQT19X3 SI ALNVYYVM SIHL uoneunsep go4 peddius ale sued juawsaoejdal pue pied aBejsod oway eq 3snu sued Buiuu40juoo uou UE uodo s ouueu 1 3ueuudinbe Jo sjueuodujoo Aug jo Una JO jeAo1dde Joud auf snu jueunjedeq searues eoiuuoe eu poled AueueM EU LO ayy puo eq Wed juauodw 09 y o JO jueuudinbe y JayHa O jueJiem ay puejxe jou I EUS jueJem siy Jepun juawdinba Jo sued sjueuoduJoo jo aeda JO jueujeose desJ AJUCIIEM SIU wo pepnjoxe ase sjexseB pue Buiqn suabeal sayy ssej6 sway ejqepuedx3 siieda Aue jo eoueuuojed oj soud uonoeJip pue uoneuruJejep jueJem pejoejuoo eq jsnuJ jueunjedeq 590 IEo1UU L eu juawsa1Be uenem siy Aq D L AO JOU aJe uoneJqi eo pue uonejejsu papnjoxa Joqe esuedxe 5 16 peoejdoJ Jo paJuredaiJ eq II diysuewWyJOM JO sjenazew ui BuiuJojuoo uou aq sued jueuoduuoo 18111 ayy Buung ueuniedeq seaiues eo1uy9a v O peuunjeJ pue p ldulo si pueo AjueJIe eu pepi oJd uonejejsur pue 40 sujuouJ xis jeuonippe ue pamolje ase juaudinba sno 390 s oym siajesg poued AjueJem Jes 1sJij ayy Buunp Jeuwo juanbasqns Aue o spueyxe uon 0ej0ud AjUeUeM aU l pasanij
47. nd 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 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 DI edge of the mesh continue rolling in d e the same direction until coil of mesh and membrane is well 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
48. nt to follow correct safety procedures when using Hybridisation Bottles Please see Appendix Il 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 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 wide
49. nts 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 increased 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
50. o 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 0 03 0 07 fl oz 1 2ml can produce dry areas on the membrane and so should be avoided e l ue Figure 5 1 Variable Axis Rotation To alter the bottle angle 1 Release the two securing screws visible on the front of the 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 position Rotisseries are available that can hold 0 5 fl oz 15ml and or 1 7 fl oz 50ml tubes Details are given in Chapter 8 Caution Bottles should be inserted into the rotisserie by sliding the bottle in sideways Care should be taken not to push the bottles too firmly into the rotisserie as the bottles will crack A Hybridisation Oven 5 1 Mesh Bottles and Caps Thermo Scientific Section 6 Cleaning and Decontamination All items which come into contact with a labelled 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 The quantity of radioactivity adhering to the mesh can be minimised by leaving the mesh in with the membrane througho
51. o 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 wet 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
52. obe 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 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 0 196 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
53. oints 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 Size 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 fo
54. om 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 Adda sufficient volume of the pre warmed first wash solution to totally immerse 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
55. or 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 A 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 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 continued Thermo Scientific Method 2 Washing ina Sandwich Box cont Other Procedures Thermo Scientific Section 3 Methodology 4 Add a sufficient volume of the pre warmed first wash solution to totally immerse the membranes Replace the lid and shake on the linear motion shaking platform in the Maxi 14 Hybridisation Oven at the required temperature for the specified time period 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 pre warmed second wash solution Replace the lid and shake at the required temperature for the required time period Step 5 should be
56. phoresis 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 specialised 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
57. ply pipette the probe into the pre hybridisation fluid in the bottle Take care to avoid pipetting the probe directly onto 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 hybridisation solution 5 Place the bottle back in the Oven switch on the rotisserie and allow it to hybridise for the required time period Thermo Scientific Hybridisation Oven 3 3 Section 3 Methodology 3 4 Washing Method 1 Washing in Hybridisation Bottles Method 2 Washing ina Sandwich Box Hybridisation Oven There are two methods of washing bottle or box Details follow Remove the bottle from the Oven Pour off the hybridisation fluid 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 A 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 f
58. quation see Meinkoth and Wahl 1984 Tm 81 5 C 16 6 logM 0 41 G C 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 formamide 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 Temperature Monovalentation Summary of Conditions Affecting Hybridisation Temperature The Hybridisation temperature is on
59. r 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 LT Agarose gel II Ee 77 Gel support 3MM VVhatman filter paper vvick Transfer 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 HRA 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
60. r least 30 minutes replacing with fresh solution after 15 minutes 5 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 advantages 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
61. re as follows 10ml Solution Volume Final Concentration 20 x SSPE or SSC 6 x SSPE SSC 100 x Denhardt s 0 5ml 5xDenhardts reagent 10 SDS 0 5 SDS Add denatured salmon sperm DNA to 50ug ml Thermo Scientific May 2003 Issue 7 30 11 Prehybridisation Buffer for Northern Blots Prepare as follows 10ml 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 e
62. 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 Membranes 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 a
63. ridisation Oven 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 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 occasions the mesh and membrane can become tightly rolled up in the bottle This occurs if the mesh is loaded incorrectly see Figure 3 1 in Section 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 considered 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
64. riments 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 10096 of BETA radiation emissions of a 1 000 pCi P 32 source However users should observe precautions due to induced X ray phenomenon Bremsstrahlung occurring depending on the type s of radionuclides used A The Maxi 14 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 7 fl oz 200ml of liquid All Ovens are calibrated by matching the thermistor temperature read out seen on the LCD display to that actually measured in fluid contained within a Hybridisation Bottle as it rotates The thermometer used inside the calibration bottle is traceable to national standards This calibration method ensures all experiments carried out using the rotisserie meet our temperature accuracy specification of 1 C and indeed due to the rotisserie 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 therefore the temperature of objects vessels which are not placed in the rotisserie will vary from that which is displayed Hybridisa
65. s 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 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 remov
66. sation Oven 3 5 Thermo Scientific Section 4 Shaking Incubations It is possible to perform shaking incubations with the Maxi 14 Hybridisation Oven l Orbital Motion For orbital shaking select the shaking platform with the round plastic central piece and four pegs Ensure that the label faces towards the front of the Oven The hole in the central plastic piece fits on the drive pin and the four pegs locate in the outer channels of the four plastic support pillars in the base of the unit Linear Motion For linear shaking motion select the shaking platform with the rectangular central piece and four metal boards The hole in the central piece fits on to the drive pin in the base of the unit with the hole pointing from front to back After the appropriate platform has been fitted switch on the shaking platform and select the desired speed Hybridisation Oven 4 1 1 7 amp 0 5 fl oz Tube Thermo Scientific Rotisseries Section5 Rotisseries All Maxi 14 Hybridisation Ovens are equipped with the 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 3 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 v oz 2 5ml T
67. t SSPE SSC and replace with pre warmed pre hybridisation buffer 6 Place the bottle in the Oven so that it rotates in the Oven in the same direction it was unrolled in Step 4 Figure 3 1 Insertion 3 2 Hybridisation Oven Thermo Scientific Section 3 Methodology Pre Hybridisation 1 When 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 51 fl oz 10 20ml of 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 1 Denature the purified probe by boiling for five 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 2 Remove the bottle from the Maxi 14 Hybridisation Oven and unscrew the cap 3 Ifthe same buffer is to be used for hybridisation as for pre hybridisation sim
68. tches to the left operate the shaking platform and the rotisserie The rotisserie and shaking platform switches cannot be operated without first turning on the power switch Instructions on how to set up the shaking platform are given in Chapter 4 The Maxi 14 Hybridisation Oven is fitted with a Type II temperature controller Follow the instructions below to set the temperature Type II temperature controllers have 3 buttons marked A A and W underneath the temperature display To reset the temperature press the key marked X while pressing either the up or down arrow key until the desired temperature is shown in the display then release the X button If the button marked is pressed and released the temperature display will show the programmed temperature setpoint The warm up time for the Oven varies according to the load and the ambient temperature When first turning on the Oven allow approximately one hour for the temperature to stabilise Solutions should be pre warmed to the required temperature to minimise sample stabilisation time 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 Maxi 14 Hybridisation Oven ensure a gap of at least 2 in 5cm is maintained between the sides of the Oven and any other equipment or object
69. te 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 protect 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 W 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
70. 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 handled wearing 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 fo
71. ther 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 counts per minute 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 Thermo Scientific Summary of Factors Resulting in High Backgrounds Thermo Scientific Section 10 Appendix Hybridisation solutions 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 pre hybridisation or blocking agents in 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 SD
72. tion Oven 1 1 Section 1 Introduction 1 2 Oven Temperature Monitoring cont Hybridisation Oven The Maxi 14 Hybridisation Oven is supplied with either two or four hybridisation bottles These high quality leak proof containers can be used at temperatures up to 70 C for part numbers see Chapter 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 The pressure should be relieved by simply loosening and then retightening the bottle cap at approximately 60 C Caution Dropping or over tightening lids can cause cracking and failure Lids should be inspected prior to each use A Note Remember to always wear gloves when handling the bottle and to use the bottle gripper provided see Appendix II for full instructions on Bottle Care A Finally the Maxi 14 Hybridisation Oven has been designed for reliability and for ease of maintenance Hence the rotisserie drip tray shaking platform can be easily removed for cleaning Thermo offers full service and technical support for all its products Thermo Scientific Section 2 Unpacking and Installation Maxi 14 Oven Packing List Maxi 14 Oven Orbital platform Power cable Linear platform 1 4 35mm rotisserie Leveling feet Drip tray Hybridisation Guide 4 medium sized bottles Bottle gripper 1 pack mesh If any item is missing or damaged contact the Technical Services
73. ut 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 contaminated it will be necessary to leave it to decontaminate in Decon 90 for a longer time period 5 Mesh should be allowed to dry flat between uses The hybridisation bottles and caps can be decontaminated by the following procedure 1 Soak the bottles and caps in a diluted 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 Hybridisation Oven 6 1 Section 6 Cleaning and Decontamination Oven Interior Shaking Stainless steel drip trays are provided with all Ovens and are intended to Platform contain spillages in the event of an accident These together with the stainless steel surfaces of the Ovens and the shak

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