PALM Protocols – DNA handling
Contents
1. Carl Zeiss Microlmaging PALM Protocols DNA at laleliial gt We make it visible PALM Protocols DNA handling Non contact Laser Capture Microdissection Carl Zeiss Microlmaging Location Munich Germany Content LO O WW O U 10 10 11 11 12 12 12 12 13 13 13 13 13 14 14 14 15 15 16 16 16 16 17 1 7 18 19 19 20 20 20 21 ZZ 22 22 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 Parattin Embedded FFPE sections Cytospins Blood and tissue smear Staining procedures Storage Formalin Fixed Paraffin Embedded FFPE sections Frozen sections Cresyl Violet Hematoxylin Eosin HE Methyl Green Methylene Blue Toluidine Blue Nuclear Fast Red 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 lifted2. hesion of the section to the PEN membrane PALM Protocols DNA Handling Hematoxylin Eosin HE HE staining is used routinely in most histo logical laboratories The nuclei are stained blue the cytoplasm pink red Staining Procedure 1 after fixation 2 min 70 Ethanol dip slide 5 6 times in distilled water 2 stain 1 2 minutes in Mayer s Hematoxy lin solution e g SIGMA MHS 32 3 rinse 1 2 min in distilled water or blueing solution 4 stain 10 seconds in Eosin Y e g SIGMA HT110 2 32 5 perform a quick increasing ethanol series 70 96 100 6 air dry shortly 1 2 min Toluidine Blue The nuclei are stained dark blue the cytoplasm lighter blue 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 m 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 SIGM
3. 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 logical information of the samples due smoothens the rough tissue surface resul to enhanced color balance and contrast ting in enhanced morphology 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 415101 4400 250 500 pl Liquid Cover Glass Order No 415190 9020 000 AdhesiveCap opaque Order No 415101 4400 240 200 pl PALM Protocols DNA Handling Collection devices AdhesiveCap Other microfuge tubes The intention of AdhesiveCap is to allow Other commercially available plasticware LCM Laser Capture Microdissection can be used too without applying any capturing liquid e g ABgene AB 0350 0 5 ml tubes into the caps prior to LCM This minimizes nuclease activity Beside the quick relocation of the lifted AmpliGrid AG480F samples inside the cap due to instant immobilization there is no risk of evapo Using the SlideCollector 48 in conjunction ration and crystal formation of the buffer with AmpliGrid technology from Advalytix during extended sp
4. 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 OIAampeE Micro Kit for isolation of genomic DNA from Laser Microdissected tissues 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 2 18h with occasional agitation Note The time necessary for complete Proteinase K digestion depends on the kind and the amount of collected material Add 25 ul Buffer ATL and 50 ul Buffer AL close the lid and mix by pulse vortexing for 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 Brietly centrifuge the 0 2 ml tube to remove drops from 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 Eppendorg 5415D 8000 rpm for 1 min Place the QlAamp MinElute Column i
5. 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 can be attached to a metal frame FrameSlide PET The frame structure of FrameSlide PET is resistant to microwave treatment or pressure cooking The special bonding is inert and adapted to heat treatment up to 95 C so that the membrane does not ruffle during the heating process If you need fur
6. membrane it is advisable to irradiate 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 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 10 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
7. pl On chip Single Cell Analysis for PALM MicroBeam AdhesiveCap opaque Order No 415101 4400 240 200 pl AdhesiveCap clear Order No 415101 4400 255 500 pl AdhesiveCap clear Order No 415101 4400 245 200 pl 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 check the right position of the correction collar see page 6 perform non contact LCM of selected cells after 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 h with occasional agitation centrifuge the tube at full speed for 5 min Hettich Centrifuge Universal 32R 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 QlAamp DNA Micro Kit 56304 page 21 step 4 can be attached 17 PALM Protocols DNA Handling Collection procedures Wet collection other microfuge tubes Note The time necessary tor complete Proteinase K digestion depends on
8. samples Downstream Applications DNA isolation from FFPE sections DNA isolation from frozen sections 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 Introduction Some remarks on DNA Human Genome Project has shown new insights to poorly understood biological phenomena 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 these results include biomedical and biological research toxicology drug design forensics animal and plant genetics and many others In all special fields the methods for molecular testing must be able to determine and analyze DNA sequences accurately and rapidly and whenever possible easy to use highly automated and minimized Beside these requirements on the molecular genetic method the step before generation of a 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 sample preparation PALM Protocols DNA Handling Preparation of slides Samples on MembraneSlide MembranesSlide is a glass slide covered with a membrane on one side This membrane is easily
9. 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 programming the cycler 2 Prepare a reaction mix according to setup Reaction Setup 10x Buffer 5 ul dNTP 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 ul 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 denaturation 30 sec 95 C 30 sec 50 C 30 sec 72 C annealing extension number of cycles 35 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 second sub sequent nested PCR in a capillary cycler or in a 96 well block cycler 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 vo
10. A T 0394 3 rinse in distilled water 4 perform a quick increasing ethanol series 70 96 100 5 air dry shortly 1 2 min 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 81963 3 rinse in distilled water 4 air dry shortly 1 2 min 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 For LCM embedding and glass covering of the specimen is inapplicable Thus the rough open surface of the section material often results in impaired view of morphology Diffusor AdhesiveCap as well as Liquid Cover Glass are comparable to the 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 up 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
11. Is easily done by dipping the slide 5 6 times in water If the sections are stained In 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 Parattin will reduce laser efficiency some times completely inhibiting cutting and lifting IT you are working with unstained sections it is therefore very important to remove the paraffin before laser cutting and lifting 1 mm MembraneSlides can be handled like normal glass slides Deparaffination Procedure 1 Xylene 5 minutes 2 times 2 minutes minimum 2 Ethanol 100 1 minute 3 Ethanol 96 1 minute 4 Ethanol 70 1 minute Note The thin 0 17 mm MembraneSlides are not as resistant against organic solvents and should only be handled according to a minimal procedure Especially longer treat ment in xylene will resolve the glue holding the membrane to the slide PALM Protocols DNA Handling Cytospins Blood and tissue smear Cytospins can be prepare
12. d on glass slides Distribute a drop of peripheral blood or on MembranesSlides or material of a smear over the slide After centrifugation in a cytocentrifuge let Be careful to avoid injuries in the mem the cells air dry at room temperature brane which would 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 PALM Laboratories we usually perform the Cresyl Violet or Hematoxylin Eosin HE Staining Storage Stained slides can be used immediately or stored If the slides have to be stored in a freezer before LCM the slides should be frozen in a tightly sealed container e g two slides back to back in a 50 ml Falcon tube to avoid exce
13. ecimen harvesting enables working in a higher throughput For more details and handling please see LCM 48 samples simultaneously also AdhesiveCap product information The AmpliGrid technology allows DNA analysis in an extremely low volume 1 ul Note CZMI recommends AdhesiveCap directly on chip as a collection device Please see the brochure Microdissection from Carl Zeiss On Chip Single Cell Analysis for PALM MicroBeam Contemporary techniques in molecular biology have begun to solve a vast number of previously unanswered questions 5 ta x However the inherent reliability of an analytical method is iz always limited by the quality of the available starting material PALM MicroBeam from Carl Zeiss enables recovery of With the AmpliGrid technology from Advalytix DNA ampli arting material from tissue preparations as well fication and cycle sequencing are possible in an extremely as from cell cultures in vitro first step Using the PALM low volume reaction formai boMover high throughput capture device in unique The reaction is carried out conjunction with the AmpliGrid slide it is possible to control the template source secon o 48 discrete reaction sites t on chip by using single cells as d step The combination of both technologies e ors to the stu For Single Cell Genotyping Sequencing and Expression Analysis Reliable Safe Reproducible AdhesiveCap opaque Order No 415101 4400 250 500
14. es Microdissection from Carl Zeiss in Life Sciences For questions comments or protocol requests please contact PALM Laboratories Email palm labs zeiss de Service Line 49 0 89 90 9000 900 24 RNA PALM Protocols DNA Handling 25 Carl Zeiss Microlmaging GmbH Location Munich Phone 49 0 89 90 9000 800 Fax 49 0 89 90 9000 820 E Mail palm labs zeiss de www zeiss de microdissection We make itvisible PALM Protocol DNA handling November 2008
15. exing for 15 sec 7 incubate the tube at 56 C for 2 18h with occasional agitation 8 centrifuge the tube at full speed for 5 min Hettich Centrifuge Universal 32R 9 final heating step at 90 C for 10 minto inactivate Proteinase K 10 centrifuge the tube at full speed for 5 min Hettich Centrifuge Universal 32R PALM Protocols DNA Handling Wet collection onto Capture check looking into Slide48 AmpliGrid AG480F the cap to see the lifted samples A preloading of 48 ReactionSites of the To control and document the efficiency of AmpliGrid with 1 ul liquid e g 1 Glycerol lifting it is possible to have a look into the in water enables elongation of the working collection device e g microfuge cap with time and increases adhesion of the captured the 5x 10x 20x 40x and 63x objectives samples The LCM process onto 48 reaction sites can be operated automatically and is By using the software function go to controlled by PALM RoboSoftware checkpoint the slide is moved out of Using Slide48 technology DNA amplification the light path and the objective litted for doesn t require any template transfer and looking inside preparation Analysis PCR cycle sequencing can be performed on the same reaction site of the AmpliGrid AG480F see page 23 Low volume PCR 1 ul in a Eppendorf MasterCycler SlideCollector 48 with AmpliGrid AG480F PALM Protocols DNA Handling Downstream Applications DNA isolation from fr
16. lene based or water soluble the whole slide should be completely submerged in the respective solvent 1 standing up in a glass jar filled with either pure xylene or warm water 30 50 80 2 time needed for the coverslip to swim off may range from hours to days 3 air dry the slide Note it 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 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 Poly L Lysine treatment To ensure nuclease free MembraneSlides 1mm or glass slides heat slides at 180 C in a drying cabinet for 4 hours to completely inactivate nucleases Note The thin 0 17 mm MembraneSlides are not resistant against heat Use other treatments to remove nucleases UV treatment To overcome the hydrophobic nature of the
17. lume 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 Mix the individual so lutions Keep samples on ice during reaction setup or while programming the cycler 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 ul dNTP Mix 2 5 uM each 0 1 ul distilled water PCR clean 0 6 ul Total reaction volume 1 0 ul 3 Mix the reaction mix and dispense 1 ul to each microdissected cell on the 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 DU sec 2 C 10 min 72 C annealing extension number of cycles 32 Depending on the experiment a seguencing reaction on an Eppendorf Mastercycler with capillary electrophoresis analysis or a check of the specificity of the PCR product s by gel electrophoresis can be attached 23 PALM Protocols DNA Handling Other brochures and protocols Live cells Chromosom
18. m a single microdissected cell the high concentration as provided in 2x OuantiFast 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 OuantiFast SYBR Green PCR Master Mix 10 ul The low volume PCR 7 ul in an Eppen Primer A 10 uM 0 5 ul dorf Mastercycler allows a direct analysis Primer B 10 uM 0 5 ul without separate DNA Isolation and trans Template DNA lt 100 ng reaction fer step The method offers the advantage distilled water PCR clean variable of the combination of LCM and low volume Total reaction volume 20 ul 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 32 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 ul in an Eppendorf Mastercycler The input of
19. n 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 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 flow through Repeat step 8 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 full speed 20 000 rpm x g 14 000 rpm 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 i
20. ozen sections DNA isolation from FFPE sections 20 Deparaffination and staining are done according to standard procedures for slides please see page 10 12 Note Proteinase K digestion step is essential for formalin fixed samples The time necessary for optimal Proteinase K digestion depends on many factors like tissue type fixation procedure or element size 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 digestion should be applied with any extraction procedure and material For subsequent DNA extraction from FFPE sections PALM Laboratories prefer the QIA amp DNA Micro Kit 56304 please see DNA isolation from frozen sections To capture microdissected samples trom frozen sections 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 QIAamp DNA Micro Kit is designed for use of small amounts of tissue The subsequently described protocol is suitable even Tor single cells Note Incubating the QlAamp MinElute Column loaded with water for 5 min at room temperature before centrifugation generally increases DNA yield Diluted solutions of nucleic acids e g dilution series used as standards should be stored in aliquots
21. s 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 downstream application Remember that the volume of eluate will be up to 5 ul less than volume of elution solution applied to the column Close the lid and incubate at room temperature 15 25 for 1 min Centrifuge at Tull speed 20 000 x g 14 000 rom for 1 min 21 PALM Protocols DNA Handling Downstream Applications PCR setup Standard PCR 20 ul in 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 OuantiFast SYBR Green PCR Master capillary cycler is usetul 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 fro
22. ss condensation of moisture during thawing For rethawing the container should not be opened before it is completely warmed up again to ambient temperature 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 for 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 staining is desired a reinforcement by two additional steps in 50 ethanol first before staining in cresyl violet second after the staining in cresyl violet is possible 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
23. 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 with When using unfilled routine microfuge tubes it is necessary to Till a liquid into the cap to improve the adhesion of the captured cells The detergent Igepal CA 630 in the cap turing butter 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 Collection Procedure If not going on immediately store the samples at 20 C 0 05 M EDTA pH 8 0 20 ul 1 take an autoclaved microfuge tube 1 M Tris pH 8 0 200 ul 2 pipette 3 15 ul of Capturing Buffer Igepal CA 630 SIGMA 1 3021 50 ul without Proteinase K or DNase free Proteinase K20ma ml ou water in the middle of the cap ao 4iluase den 3 put the cap tube into the collector m mu check the right position of the correction Proteinase K Solution 20 mg ml collar see page 6 jagen 19131 perform non contact LCM of selected cells Qiag 4 perform non contact LCM of selected cells Final Concentration 20 mM Tris 0 1 mM 5 centrifuge the tube at full speed for 5 min EDTA 0 5 Igepal 1 Proteinase K Hettich Centrifuge Universal 32R Always prepare a fresh mixture of 6 add 10 15 ul Capturing Buffer containing Capturing Buffer and Proteinase K Proteinase K and mix by pulse vort
24. ther information about these slides please contact E Mail palm 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 Tor 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 MembraneSlide 1 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 also see Application Note AutoLPC trom glass slide result in good quality RNA Even archival sections can be used after removing the coverslip and the mounting medium To facilitate lifting additional adhesive sub stances or Superfrost charged slides should only be applied when absolutely necessary for the attachment of poorly ad hering special material e g some brain sections or blood vessel rings In those cases higher laser energy is needed Tor lifting Depending on the applied mounting medium whether it was xy
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