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Scientific

, May 2003. Issue 7

APPENDIX I

SOLUTIONS FOR NUCLEIC ACID BLOTTING & HYBRIDISATION

PROCEDURES

20 x SSC (1 litre)

NaCl

175g

Sodium citrate

88g

Dissolve in 800ml of H

O and adjust pH to 7.0 with NaOH solution. Adjust the

volume to 1 litre with H

O and sterilise by autoclaving.

20 x SSPE (1 litre)

NaCl

175g

NaH

O 27.6g

EDTA 7.4g

Dissolve in 800ml of H

0 and adjust pH to 7.4 with NaOH solution. Adjust the

volume to 1 litre with H

O and sterilise by autoclaving.

100 x Denhardt’s Reagent

Ficoll

Polyvinyl pyrollidone

Bovine serum albumin (Fraction V) 2g

Dissolve in 100ml H

O, filter and store in aliquots at -20

Salmon Sperm DNA

Dissolve the DNA at a concentration of 1mg/ml in sterile H

O, sonicate to a length

of 200-500bp, and then stand in a boiling water bath for 10 minutes. Aliquot and
store at -20

5. Denaturing

Solution

NaCl

1.5M

NaOH 0.5M

Sterilise by autoclaving.

Содержание Shake 'n Stack 6244

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

Страница 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...

Страница 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...

Страница 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...

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

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

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

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

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

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

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

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

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

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

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

Страница 42: ......

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

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

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

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 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...

Страница 91: ......

Страница 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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