Thermo Scientific Shake 'n Stack 6244 Operating And Maintenance Manual Download Page 70 | Manualshive

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© Thermo

Scientific

, May 2003. Issue 7

17

4.

Add a 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.

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Summary of Contents for Shake 'n Stack 6244

Page 1: ...Visit us online to register your warranty www thermoscientific com labwarranty User Manual Shake n Stack Hybridisation Oven Operating and Maintenance Manual 7026240 Rev 1...

Page 2: ...0 25036 OV 368 8 16 11 New controller Release 3 ccs REV ECR ECN DATE DESCRIPTION By Preface Shake n Stack i Models covered by this manual Models Voltage Includes 6240 220 Shaking platform drip tray r...

Page 3: ...t performance s Caution All internal adjustments and maintenance must be performed by qualified service personnel s Material in this manual is for information purposes only The contents and the produc...

Page 4: ...t that has been put on the market after 13 August 2005 This product is required to comply with the European Union s Waste Electrical Electronic Equipment WEEE Directive 2012 19 EU It is marked with th...

Page 5: ...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...

Page 6: ...Up the Shaking Platform 3 1 Methodology of Hybridisation 4 1 Place Membranes in a Bottle 4 1 Insert Membranes in a Bottle 4 2 Pre Hybridisation 4 3 Hybridisation 4 3 Washing 4 4 Washing in Hybridisat...

Page 7: ...ories 8 3 Exploded Parts Drawings 8 4 Electrical Schematics 9 1 Warranty Information 10 1 Troubleshooting Guide for Nucleic Acid Hybridisations AI 1 Background Reduction General AI 1 Factors Resulting...

Page 8: ...adionuclides used s The Hybridisation Oven itself provides additional shielding In the event of a spillage within the Oven the stainless steel drip tray will contain up to 6 8 fl oz 200 ml of liquid N...

Page 9: ...x II for full instructions on Bottle Care s Finally the Shake n Stack Oven is designed for reliability and ease of maintenance The rotisserie shaking platform and drip tray can be easily removed for c...

Page 10: ...or short Hybridisation Bottles Once unpacked attach leveling feet to base and position on a flat surface Level the oven by adjusting the height of the leveling feet Once leveled the oven is ready for...

Page 11: ...switched on the display shows three dashes as the controller goes through its internal self tests which are completed within 5 seconds The display then shows the compensated temperature of the oven T...

Page 12: ...int value of 55 C minus a thermometer reading of 58 C yields a remainder of 3 C which means that the adjusted setpoint value for this protocol will be 52 C Repeat this process to verify that the therm...

Page 13: ...2 4 Shake n Stack Thermo Scientific Section 2 Unpacking and Installation...

Page 14: ...ten screws to secure back plate See Figure 2 2 Fit the rear right hand peg of tray into vertical slot at right of bracket allowing tray to temporarily rest on oven base Refer to Figure 3 Shake n Stack...

Page 15: ...drive shaft ensuring the sleeve is pushed fully onto shaft Tighten using thumb screw 4 Locate front left peg of tray into the hole at the end of the drive arm 5 Slide the rear left peg into the horizo...

Page 16: ...8 in 20 x 20 mm membranes can be hybridised in a single hybridisation bottle 5 Place 0 34 0 51 fl oz 10 15 ml SSPE SSC into a hybridisation bottle and then insert the roll in such a way that the leadi...

Page 17: ...Figure 4 2 3 Secure cap and holding bottle horizontally roll to catch the trailing edge of the mesh continue rolling in the same direction until coil of mesh and membrane is well positioned 4 Pour out...

Page 18: ...ially different than that used previously for hybridisation in bags or boxes ensure that the quantity of the probe is adjusted accordingly to maintain the correct probe concentration If this is not do...

Page 19: ...fer to the Hybridisation Guide Note All wash solutions should be pre warmed for best results Some scientists prefer to remove the membranes from the bottles and wash them all in one container Washing...

Page 20: ...ormed at ambient temperature i Depurination 0 25 M HCI 10 minutes ii Denaturation 1 5 M NaCI 0 5 M NaOH 30 minutes iii Neutralisation 1 5 M NaCI 0 5 M Tris CI pH 7 2 30 minutes 2 Pre washing of Filter...

Page 21: ...4 6 Shake n Stack Thermo Scientific Section 4 Methodology of Hybridisation...

Page 22: ...and active hybridisation or washing stage and can allow probe volumes to be reduced to as low as 0 07 0 17 fl oz 2 5 ml To achieve the optimum active wave conditions requires adjustment of the rotisse...

Page 23: ...vailable that can hold 0 5 fl oz 15 ml and or 1 7 fl oz 50 ml disposable tubes Details are given in Section 8 Tubes should be inserted into the rotisseries by sliding the tube sideways into the rotiss...

Page 24: ...en 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 If the mesh is still contamina...

Page 25: ...hermo ovens and are intended to contain spillages in the event of an accident These together with the stainless steel surfaces of the ovens and the shaking platform can be decontaminated by wiping cle...

Page 26: ...ase refer to the Hybridisation Guide for guidelines on the use of non radioactive systems Warning The Shake n Stack Oven has not been designed for use with hazardous or volatile chemicals with low fla...

Page 27: ...7 2 Shake n Stack Thermo Scientific Section 7 Use of Radioactive Probes...

Page 28: ...ong medium short bottles Material Stainless steel shaft Delrin plastic rotisserie wheels Variable axis 0 15 Shaker Speed 4 10 rpm Maximum Weight Capacity 2 2 lb 1 kg Max Load Dimensions 9 8 W x 7 1 H...

Page 29: ...1 4 in 35mm Rotisserie Models 6240 6241 Ordering Information Shake n Stack 110 V Model 6241 220 V Model 6240 Includes shaking platform drip tray and Delrin plastic rotisserie 110 V Model 6243 220 V Mo...

Page 30: ...idisation Ovens P N 222044 Holds up to 1 5 x 1 7 fl oz 44 x 50 ml tubes Shaking Platform P N 222000 Hybridisation Bottles Extra long bottle 2 8 x 11 8 in 70 x 300 mm P N 110094 Long bottle 1 4 x 11 8...

Page 31: ...8 4 Shake n Stack Thermo Scientific Section 8 Technical Specifications...

Page 32: ...Shake n Stack 8 5 Thermo Scientific Section 8 Technical Specifications...

Page 33: ...8 6 Shake n Stack Thermo Scientific Section 8 Technical Specifications...

Page 34: ...Shake n Stack 8 7 Thermo Scientific Section 8 Technical Specifications...

Page 35: ...8 8 Shake n Stack Thermo Scientific Section 8 Technical Specifications...

Page 36: ...Shake n Stack 8 9 Thermo Scientific Section 8 Technical Specifications...

Page 37: ...8 10 Shake n Stack Thermo Scientific Section 8 Technical Specifications...

Page 38: ...Shake n Stack 9 1 Thermo Scientific Section 9 Electrical Schematics...

Page 39: ...9 2 Shake n Stack Thermo Scientific Section 9 Electrical Schematics...

Page 40: ...Shake n Stack 9 3 Thermo Scientific Section 9 Electrical Schematics...

Page 41: ...9 4 Shake n Stack Thermo Scientific Section 9 Electrical Schematics...

Page 42: ......

Page 43: ...ength of the bottles and that there is sufficient probe solution to cover the entire membrane On occasion the mesh and membrane can become tightly rolled up in the bottle This occurs if the mesh is lo...

Page 44: ...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...

Page 45: ...e salt concentration ii Increase temperature iii Increase concentration of SDS iv Increase wash times 6 Membranes drying out This may often be the cause of an apparent overlap problem and may result f...

Page 46: ...ces the solvent volume and has the same effect 5 No probe homology 6 Double stranded DNA probe was not denatured see standard protocols Alternatively probe degraded This is more likely to occur when u...

Page 47: ...is important to check your bottles regularly for chips stress fractures and cracks If these occur discard the bottle Ensure bottles are stored either in a suitable rack or with caps replaced between e...

Page 48: ...ond the original warranty period The Technical Services Department must give prior approval for return of any components or equipment At Thermo s option all non conforming parts must be returned to Th...

Page 49: ...acement or repair of components parts or equipment under this warranty shall not extend the warranty to either the equipment or to the component part beyond the original warranty period The Technical...

Page 50: ...A 8 Shake n Stack Thermo Scientific...

Page 51: ...HYBRIDISATION GUIDE USER INSTRUCTION MANUAL Manual 7222060 Rev 0...

Page 52: ...HYBRIDISATION PROCEDURES 9 Southern Blot DNA Hybridisations 9 Northern Blot RNA Hybridisation 11 Notes for Nucleic Acid Hybridisations using the Thermo Range of Equipment 12 Placing Membranes in a Bot...

Page 53: ...ower than Expected 28 APPENDIX I SOLUTIONS FOR NUCLEIC ACID BLOTTING HYBRIDISATION PROCEDURES 29 APPENDIX II FACTORS AFFECTING STRINGENCY OF HYBRIDISATION REACTIONS 32 Effect of Temperature Salt Conce...

Page 54: ...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 immobi...

Page 55: ...following laboratory manuals in addition to literature cited in the references Fritsch J Maniatis T 1989 Molecular Cloning A Laboratory Manual 2nd Edition Sambrook Cold Spring Harbour Laboratory Press...

Page 56: ...er s instructions to operate apparatus correctly If samples are spotted manually apply 0 5 1 0 l aliquots and allow to dry between applications to prevent excessive spreading 5 Dismantle the apparatus...

Page 57: ...should also be marked with orientation points 4 The replica membranes are then placed colony side up on to fresh agar plates containing the appropriate selective antibiotic and incubated at 37 C unti...

Page 58: ...at least 30 seconds Orientation points should be marked with a sterile needle Further replicas may be prepared by leaving the Hybridisation membrane for progressively longer periods of time on the su...

Page 59: ...ults 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 auto...

Page 60: ...f 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...

Page 61: ...steps to facilitate transfer of large RNA molecules as follows 50mM NaOH 10mM NaCl 30 minutes 100mM Tris HCl Ph7 5 30 minutes Gentle agitation of the gel is essential to prevent damage to the gel duri...

Page 62: ...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...

Page 63: ...0 1 SDS at 65 C 1 x 30 minutes with 1 x SSPE SSC 0 1 SDS at 65 C 1 x 10 minutes with 0 1 x SSPE SSC 0 1 SDS at 65 C The final wash is a high stringency wash Use of a hand held monitor to give an indi...

Page 64: ...if in a Hybridisation bottle 10 20ml for a large bottle and 5 10ml for a small bottle 4 Denature the labelled probe by heating to 100 C and incubating for 5 minutes Chill on ice and add to the prehyb...

Page 65: ...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...

Page 66: ...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 shoul...

Page 67: ...and then insert the roll centrally 3 Secure cap and holding bottle horizontally roll to catch the trailing edge of the mesh continue rolling in the same direction until coil of mesh and membrane is we...

Page 68: ...ifferent 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 b...

Page 69: ...sh step 4 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...

Page 70: ...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 Repl...

Page 71: ...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 activi...

Page 72: ...ligonucleotide 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 oli...

Page 73: ...femtogram levels by using a chemiluminescent reaction The B ehringer DIG system with the same alkaline phosphatase conjugate will generate light with the chemiluminescent substrate AMPPD The resultant...

Page 74: ...bridisation membrane below e One piece of nylon mesh This procedure prevents high backgrounds Mesh and dummy membrane are reusable after washing in distilled water 3 Roll sandwich ensuring no air bubb...

Page 75: ...in 50 100ml 4 x SSC 1 SDS at 68 C 2 x 15 minutes in 50 100ml 2 x SSC 0 1 SDS at 68 C 1 x 15 minutes in 50 100ml 0 1 x SSC 0 1 SDS at 68 C Alternatively remove membrane from roll and wash in plastic b...

Page 76: ...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...

Page 77: ...SDS at the Tm 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 p...

Page 78: ...e 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 ro...

Page 79: ...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...

Page 80: ...n to the membrane 5 Hybridisation and or washing conditions not stringent enough i Decrease salt concentration ii Increase temperature iii Increase concentration of SDS iv Increase wash times 6 Membra...

Page 81: ...n 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 Alternativel...

Page 82: ...g Dissolve in 800ml of H20 and adjust pH to 7 4 with NaOH solution Adjust the volume to 1 litre with H2O and sterilise by autoclaving 3 100 x Denhardt s Reagent Ficoll 2g Polyvinyl pyrollidone 2g Bovi...

Page 83: ...onised 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 9 Pre wash Solution 5 x SSC 0 5 SDS 1mM Na2EDTA 10 Prehybridisation...

Page 84: ...l 5 x Denhardt s 10 SDS 0 5ml 0 5 SDS H2O 1 5ml Add denatured salmon sperm DNA to 100 g ml NB All solutions should be prepared in clean sterile glassware using distilled water and highest quality reag...

Page 85: ...itions relating to oligonucleotide Hybridisations As an illustration in a reaction carried out in a solution of 6 x SSC and no formamide with a 50 GC rich 500bp probe the Tm is calculated to be 101 C...

Page 86: ...overcome the repulsive forces between the negatively charged strands If the temperature is increased then the stringency of Hybridisation is increased Ionic Strength Increasing the monovalent cation c...

Page 87: ...easing local probe concentration It also acts to some degree as a blocking agent Blocking Agents For example sonicated salmon sperm DNA Denhardt s reagent Blocking agents act as analogues to the probe...

Page 88: ...ant 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 betwe...

Page 89: ...ques in situ Science 196 180 5 Southern E M 1975 Detection of specific sequences among DNA fragments separated by gel electrophoresis J Mol Biol 98 503 6 Medveczky P Chang C W Oste C and Mulder C 1987...

Page 90: ...iati 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...

Page 91: ......

Page 92: ...er Scientific and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details The...

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