PALM protocols-DNA handling (Zeiss)
Contents
1. MembraneSlide is a glass slide covered with a membrane on one side This membrane is easily cut together with the sample and acts as a stabilizing support during lifting Therefore even large areas are lifted by a single laser pulse without affecting the morphological integrity of the tissue Use of MembraneSlide is especially recommen ded for isolating single cells or chromosomes as well as live cells or small organisms Carl Zeiss Microlmaging CZMI offers slides 1 mm 0 17 mm covered with Polyethylene Naphthalate PEN membrane or Polyethylene Teraphthalate PET membrane PEN membrane is highly absorptive in the UV A range which facilitates laser cutting The membrane can be used for all kind of applications MembraneSlide NF nuclease free is certified to be tree of DNase RNase and human DNA In addition to PEN MembraneSlide CZMI also offers PET membrane covered slides These slides are helpful for special processes i e fluorescence applications Even weak fluores cence signals can be detected with PET slides due to the low signal to noise ratio Regular glass slide 1 mm thick gt 1 thin slide 0 17 mm thick gt dot DuplexDish and FrameSlide gt between dot and 0 Alternatively the PET membrane attached to a metal frame FrameSlide PET is also available The structure of FrameSlide PET is resistant to microwave treatment or pressure cooking The special bonding is inert and adapted to heat tr2. On chip Single Cell Analysis for PALM MicroBeam Collection procedures Dry collection procedure Please have a look into the PALM MicroBeam user manual Dry collection AdhesiveCap Note CZMI recommends AdhesiveCap as collection device for most experiments 1 U N Ul put the AdhesiveCap into the collector and check the right position of the correction collar see page 6 perform non contact LCM of selected cells alter LCM add 15 ul lysis buffer to the Sample inside the cap QlAamp DNA Micro Kit 56304 add 10 ul Proteinase K 20 mg ml and mix by pulse vortexing for 15 sec place the tube in an upside down position in an incubator at 56 C for 2 18 with occasional agitation centrifuge the tube at 10000 rcf for 5 min Tabletop Microcentrifuge If not going on immediately store the Samples at 20 C Note The time necessary tor complete Proteinase K digestion depends on the kind and the amount of collected material After the Proteinase K digest the regular procedure of the QlAamp DNA Micro Kit 56304 page 21 step 4 can be attached Note Please do not use any water bath for the upside down incubation 17 PALM Protocols DNA Handling Collection procedures Wet collection other microfuge tubes When using unfilled routine microfuge tubes it is necessary to add a liquid into the cap to facilitate the adhesion of the cap tur
3. logical information of the samples due smoothens the rough tissue surface resul to enhanced color balance and contrast ting in enhanced morphology after drying which makes the view comparable to For more details and handling please see those of coverslipped tissue sections Liquid Cover Glass product information Two different microfuge tube sizes 200 ul 500 ul with these filled caps are available from CZMI For more details and handling please see AdhesiveCap product information AdhesiveCap opaque Order No 415190 9201 000 500 pl Liquid Cover Glass Order No 415190 9020 000 AdhesiveCap opaque Order No 415190 9181 000 200 pl 15 PALM Protocols DNA Handling Collection devices AdhesiveCap The intention of AdhesiveCap is to allow LCM Laser Capture Microdissection without applying any capturing liquid into the caps prior to LCM This minimizes the risk of nuclease activity Beside the quick relocation of the lifted samples inside the cap due to instant immobilization there is no risk of evapo ration and crystal formation of the buffer during extended specimen harvesting For more details and handling please see also AdhesiveCap product information AdhesiveCap opaque Order No 415190 9201 000 500 pl AdhesiveCap opaque Order No 415190 9181 000 200 ul AdhesiveCap clear Order No 415190 9211 000 500 pl AdhesiveCap clear Order No 415190 9191 000 200 ul Other microfuge tubes Other
4. stain 1 2 minutes in Mayer s Hematoxy lin solution e g SIGMA MHS 32 This short staining procedure colors the nu clei violet and the cytoplasm weak violet Staining Procedure 1 after fixation 2 min 70 Ethanol dip slide for 30 sec into 1 cresyl violet acetate solution 2 remove excess stain on absorbent surface 3 dip into 70 Ethanol 4 dip into 100 Ethanol 5 air dry shortly 1 2 min Dissolve solid cresyl violet acetate e g ALDRICH 86 098 0 at a concen tration of 1 w v in 50 EtOH at room temperature with agitation stirring tor several hours to overnight Filter the staining solution before use to remove unsolubilized powder Sometimes Lot to Lot variations in the purchased cresyl violet powder can lead to weaker staining results if the dye content is below 75 Note In most cases this cresyl violet staining procedure will be sufficient for cell identiti cation If an enhancement of the intensity is desired a reinforcement by two additional steps in 50 ethanol is possible first before staining in cresyl violet second after the staining in cresyl violet Ambion offers the LCM Staining Kit 1935 which also contains a cresyl violet dye When using this kit we strongly recommend to omit the final xylene step of the Ambion instruction manual because xylene makes the tissue very brittle and reduces the ad hesion of the section to the PEN membrane PALM Protocols DNA Handling Tol
5. whether it is soluble in xylene or water the whole slide should be completely submerged in the respective solvent standing up in a glass jar filled with either pure xylene or warm water 30 5020 2 time needed for the coverslip to swim off may range trom hours to days 3 gentle movement of the jar may speed up the process 4 air dry the slide after removal Note is very important NOT to use any force to push off the coverslip because this might damage the section Wait till it falls off by itself The necessary time depends on the age of the sample and the dryness of the mounting medium Fresh slides only days old can be decover slipped much faster From the dry glass slide sample material can be lifted directly by AutoLPC function of PALM RoboSoftware PALM Protocols DNA Handling Treatment of slides Slides are shipped without any pretreatment To remove potentially contaminating nucleases and DNA MembraneSlides and glass slides can be treated in the same way MembraneSlide NF nuclease free is certified to be free of DNase RNase and human DNA Treatments to remove nucleases and contaminating DNA are therefore not necessary using these slides Heat treatment To ensure nuclease free Membraneslides heat slides at 180 C in a drying cabinet for 4 hours to completely inactivate nucleases UV treatment To overcome the hydrophobic nature of the membrane it is advisable to irra
6. Master Mix 10 The low volume PCR 1 ul in an Eppen Primer A 10 uM 0 5 ul dorf Mastercycler allows a direct analysis Primer 8 10 uM 0 5 ul without separate DNA Isolation and trans Template DNA lt 100 ng reaction fer step This method offers the advantage distilled water PCR clean variable of the combination of LCM and low volume Total reaction volume 20 PCR on the same slide 3 Mix the reaction mix thoroughly and dispense appropriate volumes into PCR capillaries 4 Add template DNA lt 100 ng reaction to the individual capillaries containing the reaction mix 5 Program the cycler according to conditions Capillary Cycler conditions exemplary Step Time Temp Ramp rate PCR initial activation 5min 95 C fast mode Two step cycling denaturation 10 sec 95 C fast mode combined annealing extension 30sec 60 C fast mode number of cycles 40 6 Place the PCR capillaries in the cycler and start the cycling program 7 Optional Check the specificity of the PCR product s by agarose gel electrophoresis PALM Protocols DNA Handling High volume PCR 50 ul in a 96 well block cycler Low volume PCR 1 pl in an Eppendorf Mastercycler The input of the whole eluate 20 ul to the PCR reaction mix requires an increased total reaction volume of 50 ul PCR Procedure 1 Thaw PCR buffer dNTPs template DNA primers and water Mix the individual solu tions Keep samples on ice during reaction setup or while prog
7. commercially available plasticware can be used too e g ABgene AB 0350 0 5 ml tubes AmpliGrid AG480F Using the SlideCollector 48 in conjunction with AmpliGrid technology trom Advalytix enables working in a higher throughput LCM 48 samples simultaneously The AmpliGrid technology allows DNA analysis in an extremely low volume 1 ul directly on chip Please see the brochure labs zeiss de Microdissection from Carl Zeiss On Chip Single Call Anslysia for PALM MioroBeam Contemporary techniques In molecular biology have begun to solve a vast number of previously unanswered questions However the inherent reliability of an analytical method 5 always limited by the quality of the available starting material PURITY PALM MacroBeam from Carl Zeiss enables recovery of With the 0 1 from Adhralytiz DMA anapi maera from Issue preparat ons as well fication anal oyde sequencing ase possiile in an extremely as kam cell cultures in vitro first step Using the PALM volume reaction format RoboMorer capture device in unique The acion 5 amied aut on chip by using single cells as anuncian wath the Amplicad sade its possible to comtral the template source second step reaction sies The 2081483038 of both techaniogha opera nmmr doors do the study of pesones For Single Cell Genotyping Sequencing and Expression Analysis Reliable Safe Reproducible
8. litted samples Downstream Applications DNA isolation trom FFPE sections DNA isolation from frozen sections QlAamp DNA Procedure PCR setup Standard PCR 20 ul in a capillary cycler High volume PCR 50 ul in a 96 well block cycler Low volume PCR 1 ul in an Eppendorf Mastercycler with In situ Adapter Other protocols RNA Chromosomes Live Cells Introduction Some remarks on DNA Human Genome Project has shown new insights into poorly understood biological phenomena by providing vast DNA sequencing data As a result of this expansion of genomics into human health applications the field of genomic medicine was born Genetics is playing an increasingly important role in the diagnosis monitoring and treatment of diseases Further areas that stand to benefit trom DNA results include biomedical and biological research toxicology drug design forensics animal and plant genetics and many others In all fields the methods for molecular testing must be able to determine and analyze DNA sequences accurately and rapidly Whenever possible the procedures should be easy to use highly automated and minimized in requirement of material Therefore the generation of defined sample as source for the analysis is of prime importance Non contact Laser Capture Microdissection LCM from Carl Zeiss is state of the art for precise sample preparation PALM Protocols DNA Handling Preparation of slides Samples on MembraneSlide
9. wetting the rim Close the lid and centrifuge at 6000 x g 8000 rpm for 1 min Place the QlAamp MinElute Column in a clean 2 ml collection tube and discard the collection tube containing the flow through Repeat procedure of step 8 with 500 ul Buffer AW2 this time Note Contact between the QlAamp MinElute column and the flow through should be avoided Some centrifuge rotors may vibrate upon deceleration resulting in the flow through which contains ethanol coming into contact with the QlAamp MinElute Column Take care when removing the QlAamp MinElute Column and collection tube from the rotor so that flow through does not come into contact with the QlAamp MinElute Column Centrifuge at Tull speed 20 000 x g 14 000 rom for 3 min to dry the membrane completely This step is necessary since ethanol carryover into the eluate may interfere with some downstream applications Place the QlAamp MinElute Column in a clean 1 5 ml microcentrifuge tube not provided and discard the collection tube containing the flow through Carefully open the lid of the QlAamp MinElute Column and apply 20 ul distilled water to the center of the membrane Ensure that distilled water is equilibrated to room temperature 15 25 C Dispense distilled water onto the center of the membrane to ensure complete elution of bound DNA Note QlAamp MinElute Columns provide flexibility in the choice of elution volume Choose a volume according to the requirements of the down
10. Carl Zeiss Microlmaging PALM Protocols DNA handling de EN F i 1 We make it visible PALM Protocols DNA handling Non contact Laser Capture Microdissection Carl Zeiss Microlmaging Location Munich Germany Content WO WO WW UI 10 10 11 11 12 12 12 12 13 13 13 13 13 14 14 14 15 16 16 16 16 1 14 18 19 19 20 20 20 21 22 23 23 24 Introduction Some remarks on DNA Preparation of slides Samples on MembraneSlide Samples on glass slides Archived samples removing the coverslip Treatment of slides Heat treatment UV treatment Poly L Lysine treatment Mounting samples onto slides Frozen sections Formalin Fixed Paraffin Embedded FFPE sections Cytospins Blood and tissue smear Staining procedures Formalin Fixed Paraffin Embedded FFPE sections Frozen sections Hematoxylin Eosin HE Cresyl Violet Toluidine Blue Methyl Green Methylene Blue Nuclear Fast Red Storage Non contact Laser Capture Microdissection LCM Procedure Tips to improve morphological information Diffusor CM AdhesiveCap opaque Liquid Cover Glass Collection devices AdhesiveCap Other microfuge tubes AmpliGrid AG480F Collection procedures Dry collection AdhesiveCap Wet collection other microfuge tubes Wet collection onto Slide48 AmpliGrid AG480F Capture check looking into the cap to see the
11. PALM Lal boratories A unique service of Carl Zeiss We make it visible Immunofluorescence Microdiszection from Carl Zeiss PALM User Protocols Immunofluorescence on frozen sections PALM Laboratories A unique service of Carl Zeiss We make it visible On chip Single Cell Analysis Microdissection from Carl Zeiss On Chip Single Cell Analysia for PALM MioroBeam Contemporary techniques in molecular biology have begun to solve a vast number of previously unanswered questions However the inherent reliability of an analytical method is always limited by the quality of the available starting material YOLE 0 0 high throughput capture device 8 unique The rencia 5 came aut on chip by uing sre cols second step up to 48 discrete reaction rornblaztion of iain open amr doors to the rimy of For Single Cell Genotyping Sequencing and Expression Analysis Reliable Safe Reproducible We make It visible RNA Carl Zeiss Microlmaging PALM Protocols RNA handling For questions comments or protocol requests please contact ZEISS Labs E Mail labs zeiss de Hotline 49 8990 9000 900 24 PALM Protocols DNA Handling 25 PALM Protocols DNA handling For scientific questions please contact E Mail labs zeiss de Hotline 49 8990 9000 900 www zeiss de labs August 2011 Carl Zeiss Microlmaging GmbH 07740 Jena Germany BioSciences Loc
12. ation Munich Phone 49 8990 9000 800 Telefax 49 8990 9000 820 E Mail palm info zeiss de We make it visible www zeiss de microdissection
13. d samples Add 15 ul ATL to the microdissected sample in the AdhesiveCap Add 10 ul Proteinase K and mix by pulse vortexing for 15 sec Place the 0 2 ml tube in an upside down position at 56 C in an incubator for 3 18h with occasional agitation Note The time necessary for complete Proteinase K digestion depends on the kind of collected material Especially FFPE samples must be digested longer Add 25 ul Buffer ATL and 50 ul Buffer AL close the lid and mix by pulse vortexing Tor 15 sec To ensure efficient lysis it is essential that the sample and Buffer AL are thoroughly mixed to yield a homogeneous solution Add 50 ul ethanol 96 100 close the lid and mix thoroughly by pulse vortexing for 15 sec Incubate tor 5 min at room temperature 15 25 C IF room temperature exceeds 25 C cool the ethanol on ice before adding to the tube Briefly centrifuge the 0 2 ml tube to remove drops trom the lid Carefully transfer the entire lysate to the QlAamp MinElute column without wetting the rim close the lid and centrifuge at 6000 x g e g Eppendorf 5415D 8000 rom for 1 min Place the QlAamp MinElute Column in a clean 2 ml collection tube and discard the collection tube containing the flow through If the lysate has not completely passed through the column after centrifugation centrifuge again at a higher speed until the QlAamp MinElute Column is empty Carefully open the QlAamp MinElute Column and add 500 ul Buffer AW1 without
14. diate with UV light at 254 nm for 30 minutes e g in a cell culture hood The membrane gets more hydrophilic therefore the sections paraffin as well as cryosections adhere better Positive side effects are sterilization and destruction of potentially contaminating nucleic acids Poly L Lysine treatment Additional coating of the slide with Poly L Lysine 0 1 w v e g SIGMA P8920 only will be necessary for poorly adhering materials e g brain sections and should be performed after UV treatment Distribute a drop of the solution on top of the slide Let air dry at room temperature for 2 3 minutes Avoid any leakage of the membrane as this might result in impairment of Laser Capture Microdissection PALM Protocols DNA Handling Mounting samples onto slides Formalin Fixed Paraffin Embedded FFPE sections Frozen sections Sectioning Sectioning Sections are mounted onto MembraneSlides the same way as routinely done using glass slides To allow subsequent cutting and lifting by the laser a coverslip and standard mounting medium must not be applied Freezing media like OCT or similar may be used but should be kept to a minimum and have to be removed before laser cutting Removing the tissue freezing medium If OCT or another tissue freezing medium is used it is important to remove it before Laser Microdissection because these media will interfere with laser efficiency Removing the medium Is easily
15. done by dipping the slide 5 6 times in water If the sections are stained aqueous solutions the supporting substance is normally removed automatically by the water containing steps Floating the section on warm water 40 C as well as hot plate techniques can be applied After mounting the section let the slides dry overnight in a drying oven at 56 C to im prove the adhesion of the sections to the membrane To allow laser cutting and lifting a coverslip and standard mounting medium must not be applied Archival sections with mounting medium and coverslip have to be processed as described to remove the coverslip see page 8 Deparaffination Residual paraffin will reduce laser efficiency sometimes completely inhibiting cutting and lifting If you are working with unstai ned sections it is therefore very important to remove the paraffin before laser cut ting and lifting MembraneSlides can be handled like normal glass slides Deparaffination Procedure 5 minutes 2 times 2 minutes minimum 2 Ethanol 100 1 minute 3 Ethanol 96 4 Ethanol 70 1 Xylene 1 minute 1 minute PALM Protocols DNA Handling Cytospins Blood and tissue smear Cytospins can be prepared on glass slides Distribute a drop of blood or material of a or on Membraneslides smear over the slide After centrifugation in a cytocentrituge let Be careful to avoid injuries in the mem the cells air dry at room temperature brane which wou
16. e or thickness of lifted material An overnight digestion 12 18 hours is a good starting point for optimization but shorter digestion times may be tested as well To our experience at least 3 hours di gestion should be applied with any extraction procedure and material For subsequent DNA extraction from FFPE sections ZEISS Labs prefer the QlAamp DNA Micro Kit 56304 please see DNA isolation from frozen sections To capture microdissected samples we recommend the use of AdhesiveCap For DNA isolation any procedure of choice can be used In our hands the QlAamp DNA Micro Kit 56304 combined with AdhesiveCap results in good yield and quality of DNA This QlAamp DNA Micro Kit is designed for use of small amounts of tissue The subsequently described protocol is suitable even for single cells Note For DNA elution incubating the QlAamp MinElute Column loaded with water for 5 min at room temperature before centrifugation generally increases the final DNA yield Diluted solutions of nucleic acids e g dilution series used as standards should be stored in aliquots and thawed once only We recommend storage of aliquots in siliconized tubes if possible This avoids adsorption of nucleic acids to the tube walls which would reduce the concentration of nucleic acids in solution PALM Protocols DNA Handling Applying the components of the QIAamp DNA Micro Kit for isolation of genomic DNA from Laser Microdissecte
17. eatment even in moisture or liquid so that the membrane does not ruffle during the heating process If you need further information about these slides please contact E Mail labs zeiss de When working with low magnitying objectives like 5x or 10x both regular 1 mm thick glass slides and 0 17 mm glass slides can be used To keep this flexibility for higher magnifications 20x 40x or 63x CZMI recommends using long distance objectives With those the working distance can be adap ted to the different glass slides by moving the correction collar on the objective see picture above Due to the short working distance only 0 17 mm thin cover glass slides can be used with the 100x magnifying objectives de MembraneSlide FrameSlide PET PALM Protocols DNA Handling Preparation of slides Samples on glass slides Archived samples removing the coverslip With PALM MicroBeam almost every kind of biological material can be microdissected and lifted directly from regular glass slides Even archival sections can be used after removing the coverslip and the mounting medium To facilitate lifting additional adhesive sub stances or Supertrost charged slides should only be applied when absolutely necessary for the attachment of poorly ad hering material e g some brain sections or blood vessel rings In those cases higher laser energy is needed for lifting Depending on the applied mounting medium
18. ed cells The detergent Igepal CA 630 in the capturing buffer allows to smear out a small amount of liquid in the whole cap area Note All kinds of aqueous solutions will run dry with extended working time Prearrangements Capturing Buffer 0 05 M EDTA pH 8 0 20 ul 1 M Tris pH 8 0 200 ul Igepal CA 630 SIGMA 1 3021 50 ul Proteinase K 100 ul adH O fill up to 10 ml Proteinase K 20 mg ml Qiagen 19131 Final Concentration 20 mM Tris 0 1 mM EDTA 0 5 Igepal 1 Proteinase K Always prepare a fresh mixture of Capturing Buffer and Proteinase K Note The time necessary tor complete Proteinase K digestion depends on the kind and the amount of collected material After the Proteinase K digest and the in activation step the routine downstream application of choice can be continued If not going on immediately store the samples at 20 C Collection Procedure take an autoclaved microfuge tube NO pipette 3 15 ul of Capturing Buffer without Proteinase K or DNase free water in the middle of the cap UJ put the cap tube into the collector check the right position of the correction collar see page 6 4 perform non contact LCM of selected cells 5 centrifuge the tube at 10000 rcf for 5 min Tabletop Microcentrifuge add 10 15 ul Capturing Buffer containing Proteinase K and mix by pulse vortexing for 15 sec incubate the tube at 56 C for 2 18h with occasional agitation 8 centrifuge t
19. he tube at 10000 rcf for 5 min Tabletop Microcentrifuge O N 9 final heating step at 90 C for 10 min to inactivate Proteinase K 10 centrifuge the tube at 10000 rcf for 5 min Tabletop Microcentrifuge PALM Protocols DNA Handling Capture check looking into the cap to see the lifted samples Wet collection onto Slide48 AmpliGrid AG480F A preloading of 48 ReactionSites of the AmpliGrid with 1 ul liquid e g 1 Glycerol in water enables elongation of the working time and is necessary for adhesion of the captured samples The LCM process onto 48 reaction sites can be operated automa tically and is controlled by PALM RoboSott ware To control and document the efficiency of lifting it is possible to have a look into the collection device e g microfuge cap with the 5x 10x 20x 40x and 63x objectives By using the software function go to checkpoint the slide is moved out of the light path and the objective lifted for looking inside SlideCollector 48 with AmpliGrid AG480F PALM Protocols DNA Handling Downstream Applications DNA isolation from frozen sections DNA isolation from FFPE sections 20 Deparaffination and staining are done according to standard procedures for slides please see page 10 13 Note Proteinase K digestion step is essential for formalin fixed samples The time necessary for optimal digestion depends on many factors like tissue type fixation procedur
20. ix the individual so lutions 2 Prepare a reaction mix according to setup Reaction Setup see Advalytix protocols AmpliTaq Gold 0 1 ul 10x GeneAmp Buffer with 15mM MgCl 0 1 ul Primer 5 pmol ul each 0 1 ANTP Mix 2 5 uM each 0 1 distilled water PCR clean 0 6 ul Total reaction volume 1 0 ul 3 Mix the reaction mix and dispense 1 ul to each reaction site of the AmpliGrid slide 4 Cover the PCR droplet with 5 ul of sealing solution 5 Place the loaded AmpliGrid on the Eppen dort Mastercycler 6 Program the cycler according to conditions Eppendorf Mastercycler conditions example Step Time Temp PCR initial step 10 min 95 C denaturation 40 sec 94 C 30 sec 56 C 30 sec 72 C 311111 72 C 10 min 72 C annealing extension final extension number of cycles 40 Depending on the experiment a sequencing reaction with subsequent capillary electrophoresis analysis or a check of the specificity of the PCR product s by gel electro phoresis can be attached 23 Brochures and protocols Live cells PALM Protocols DNA Handling Chromosomes Microdissection from Carl Zeiss Laser Micromanipulation in Life Sciences Contact free manipulation of live cells Wa mata it visible FISH FISH Hybridization PALM Laboratories unique service of Carl Zeiss ke it visible Microdissection from Carl Zeiss PALM User Protocols Chromosome Preparation
21. ld lead to leakage Then fix for 2 minutes in 70 ethanol and during fixation or washing steps and air dry again before staining therefore would impair the Laser Capture Microdissection process Let smears air dry shortly and fix them for 2 up to 5 minutes in 70 ethanol PALM Protocols DNA Handling Staining procedures For isolation of high quality DNA use freshly prepared autoclaved solutions Formalin Fixed Paraffin Cresyl Violet Embedded FFPE sections After deparaffination see page 10 continue with the staining procedure of your choice Most staining procedures for frozen sections can be applied for FFPE sections for recommendations see Frozen sections Frozen sections Most standard histological stainings e g HE Methyl Green Cresyl Violet Nuclear Fast Red are compatible with Subsequent DNA isolation At ZEISS Labs we usually perform the Cresyl Violet or Hematoxylin Eosin HE Staining Hematoxylin Eosin HE HE staining is used routinely in most histolo gical laboratories The nuclei are stained blue the cytoplasm pink red Staining Procedure after fixation 2 min 70 Ethanol dip slide 5 6 times in distilled water N rinse 1 2 min in distilled water or UJ blueing solution e g BBC 3900 4 stain 10 seconds in Eosin Y e g SIGMA HT110 2 32 perform a quick increasing ethanol series 70 96 100 6 air dry shortly 1 2 min Ul
22. led container e g two slides back to back in a 50 ml Falcon tube to avoid excess condensation of moisture during thawing For rethawing the container should not be opened before it is completely warmed up again to ambient temperature PALM Protocols DNA Handling Non contact Laser Capture Microdissection LCM Procedures Please additionally have a look into the PALM MicroBeam user manual Tips to improve morphological information Embedding and glass covering of the specimen is inapplicable for LCM Thus the rough open surface of the section material often results in impaired view of morphology Effects of diffusor AdhesiveCap as well as Liquid Cover Glass are comparable to the usual coverslip for enhanced visualization Diffusor CM Holders for PALM RoboMover and PALM CapMover Il are equipped with diffusors The opaque glass diffuses the incident microscope light which smoothens the harshness of contrast and depending on material and staining even minute details as nuclei and cell boundaries show Even slight differences in color become visible For more details and handling please see Diffusor CM product information PALM CombiSystem Diffusor CM Order No 415101 2100 320 PALM Protocols DNA Handling AdhesiveCap opaque Liquid Cover Glass The white opaque filling of AdhesiveCap The polymeric and low viscose Liquid Cover clearly improves visualization of morpho Glass completely embeds the tissue and
23. ramming the cycler 2 Prepare a reaction mix according to setup Reaction Setup 10x Buffer 5 ANTP Mix 2 mM each 5 ul Primer A 10 uM 1 ul Primer B 10 uM 1 ul template DNA variable Qiagen HotStarlag Polymerase 0 5 ul distilled water PCR clean variable Total reaction volume 50 3 Mix the reaction mix thoroughly and dispense appropriate volumes into PCR tubes 4 Add template DNA lt 100 ng reaction to the PCR tubes containing the reaction mix 5 Program the cycler according to conditions Block Cycler conditions exemplary Step Time Temp activation step 15 min 95 C denaturation 30 sec 95 C 30 sec 50 C 30 sec 72 C 5 min 22 C annealing extension final extension number of cycles 40 6 Place the PCR tubes in the cycler and start the cycling program 7 Optional Check the specificity of the PCR product s by agarose gel electrophoresis Depending on the experiment a subsequent nested PCR based on the first PCR product and internal primers can be attached Advalytix protocols www advalytix com images download DNA amplification and cycle sequencing for example of a single cell are possible in an extremely low volume reaction format 1 ul with the Slide48 AmpliGrid technology After lifting the cell onto the chip analysis can be performed directly on chip without any template preparation PCR Procedure 1 Thaw PCR buffer dNTPs template DNA primers and water M
24. stream application Remember that the volume of eluate will be up to 5 ul less than the volume of elution solution applied to the column Close the lid and incubate at room temperature 15 25 for 1 5 min Centrifuge at full speed 20 000 x g 14 000 rom for 1 min 21 PALM Protocols DNA Handling Downstream Applications PCR setup Standard PCR 20 ul a capillary cycler Depending on the concentration of the QuantiFast SYBR Green PCR QIAGEN 204052 in isolated DNA the suitable setup for the our hands results in exact amplification products amplification has to be selected The standard volume PCR 20 ul in a 1 Thaw 2x QuantiFast SYBR Green PCR Master capillary cycler is useful for highly concen Mix template DNA primers and water trated DNA eluates because the maximal Mix the individual solutions input of target DNA in the reaction setup 2 Prepare a reaction mix according to setup __ is limited Only 30 50 of the eluate can Due to the hot start it is not necessary to be analysed keep samples on ice during reaction setup or while programming the real time cycler For low concentrated DNA eluates e g Note We recommend starting with the Mg from a single microdissected cell the high concentration as provided in 2x QuantiFast SYBR volume PCR 50 pl in a 96 well block Green PCR Master Mix cycler is recommendable as 100 of the Reaction Setup eluate can be used for the reaction setup 2x QuantiFast SYBR Green PCR
25. uidine Blue The nuclei are stained dark blue the cytoplasm lighter blue Staining Procedure 1 after fixation 2 min 70 Ethanol dip slide 5 6 times in distilled water 2 stain 30 seconds in Toluidine Blue solution 0 1 in water SIGMA T 0394 3 rinse in distilled water 4 perform a quick increasing ethanol series 70 96 100 5 air dry shortly 1 2 min Methyl Green The nuclei are stained dark green the cytoplasm light green Staining Procedure 1 after fixation 2 min 70 Ethanol dip slide 5 6 times in distilled water 2 stain 5 minutes in Methyl Green solution DAKO 51962 3 rinse in distilled water 4 air dry shortly 1 2 min Methylene Blue The nuclei are stained dark blue Staining Procedure 1 after fixation 2 min 70 Ethanol dip slide 5 6 times in distilled water 2 stain 5 10 min in Methylene Blue solution 0 05 in water SIGMA 3191 1 2 3 rinse in distilled water 4 air dry shortly 1 2 min Nuclear Fast Red The nuclei are stained dark red the cytoplasm lighter red Staining Procedure 1 after fixation 2 min 70 Ethanol dip slide 5 6 times in distilled water 2 stain 5 to 10 minutes in Nuclear Fast Red solution DAKO 451963 3 rinse in distilled water 4 air dry shortly 1 2 min Storage Stained slides can be used immediately or stored dry If the slides are stored in a freezer before LCM the slides should be frozen in a tightly sea
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