Automated Protocol for Extract-N-Amp™ Plant PCR - Sigma
Contents
1. Reduce the aspiration and dispensing speed and or cycle times in the mixing steps It is critical for amplifying the large genomic DNA fragments Increase the number of cycles 5 10 additional cycles ata time Refer to the Technical Bulletin of Extract N Amp Plant PCR Kits Use new labware and new batch of reagents Test a reagent blank without DNA template to determine if the reagents used in extraction or PCR are contaminated XIV Contact Information Technical Service 800 325 5832 email techserv sial com Customer Service 800 325 3010 800 588 9160 www sigma aldrich com order This product is sold under license from Roche Molecular Systems Inc and Applied Biosystems Taq Antibody licensed for in vitro research use under U S Patent No 5 338 671 and 5 587 287 and corresponding patents in other countries Biomek is a registered trademark of Beckman Coulter Inc Eppendorf is a registered trademark of Eppendorf Netheler Hinz GmbH JV KTA 10 05 1 Sigma brand products are sold through Sigma Aldrich Inc Sigma Aldrich Inc warrants that its products conform to the information contained in this and other Sigma Aldrich publications Purchaser must determine the suitability of the product s for their particular use Additional terms and conditions may apply Please see reverse side of the invoice or packing slip2. TL1 AP96 P250 Barrier Tips P2 AP96 P250 Barrier Tips P3 Swap P4 96 well reservoir for the Extraction Solution P6 Lid P7 96 well PCR plate with full skirt containing tissue samples P8 96 well reservoir for Dilution Solution Page 10 of 15 B Preparing 96 plant tissue extracts for PCR It may be desired to extract DNA from 96 plant tissue samples and set up all samples for PCR in a single 96 well PCR plate Two changes need to be made in the Extract N Amp_Plant_PCRSetup method 1 Click on the Run PCR_Setup with controls step of the Extract N Amp Plant_PCRSetup method Use the drop down arrow next to File Name to select PCR_Setup no controls method 2 Update the deck layout in the Instrument Setup step of both Extract N Amp Plant_PCRSetup and PCR_Setup no controls methods as following La DNATranst Es CRPlate xtraction a iui oo Deck Position Equipment TL1 AP96 P250 Barrier Tips Sterile P2 AP96 P250 Barrier Tips Sterile P3 AP96 P20 Barrier Tips Sterile a P4 96 well reservoir for the Extraction Solution P5 Swap E P6 Lid P7 96 well PCR plate with full skirt containing tissue samples PB 96 well reservoir for Dilution Solution sts S P11 96 well PCR amplification plate seated into a plate holder P12 12 column Reservoir for PCR master mix P44 Span 8 P250 Barrier Tips sits g C PCR setup only
3. 13 Microcentrifuge tubes 1 5 ml 2 ml screw cap 24 position Eppendorf IsoTherm System Fisher 05 405 22 12 column reagent reservoir with low profile Innovative Microplates S30028 96 well reservoir with low profile and pyramidal bottom Innovative Microplates S30018 Optional 12 column reagent reservoir with high profile Innovative Microplates S30019 Optional 96 well reservoir with high profile and pyramidal bottom Innovative Microplates S30014 Thermal Cycler Thermometer Fisher 15 077 26 Page 3 of 15 V Instrument Requirements for the Biomek FX Workstation Part Description Qty Ordering Information Orbital Shaker Contact Beckman Coulter Peltier ALP Contact Beckman Coulter Multichannel Pod 96 Mandrel 200 ul Head Contact Beckman Coulter Span 8 Pod 1 ml Syringe Contact Beckman Coulter Gripper Contact Beckman Coulter Tip Loader Contact Beckman Coulter Span 8 Tip Trash Span 8 Tip Wash Standard Passive ALPs One by Three Standard Passive ALPs One by One AP96 P250 Barrier Tips Sterile AP96 P20 Barrier Tips Sterile Span 8 P250 Barrier Tips Sterile Span 8 P20 Barrier Tips Sterile Contact Beckman Coulter Contact Beckman Coulter Contact Beckman Coulter Contact Beckman Coulter BK717253 Beckman Coulter BK717256 Beckman Coulter BK379503 Beckman Coulter BK379506 Beckman Coulter AG J 2 Ed Vi Temperature Control Device Watlow Setup Prior to
4. Tissue extracts may be subjected to additional amplifications The PCR_Setup with controls or PCR_Setup no controls method described in Section IX may be used for this purpose D Use of a different PCR plate The automated method was created using the 96 well PCR amplification plates with half skirt from ABgene Other PCR plates including 384 well plates may be used in this method but may require the creation of a new labware in the Biomek software E PCR setup using multiple primer sets To amplify genomic DNA from the tissue extract with different primer sets primers can be added to microfuge tubes and placed on the 24 position tube racks or added to the PCR ReadyMix and placed on different columns of 12 column reservoir S30028 Additional steps will need to be added to the corresponding PCR_Setup method to account for the primer addition or aspirating PCR master mix from a different column position Page 11 of 15 XII Performance Characteristics PCR Analysis of Tomato Leaf Tissue Samples M 12 34 567 8 91011 M 4234567891011 M se es es es es es es ee es ee es ee oe am Universal Chloroplast ee ed Universal Chloroplast eee ee es we ee oe D M es e oD ee ee ee a Universal Chloroplast Universal Chloroplast Figure 1 DNA was extracted from 88 Tomato leaf samples The 96 well plate was processed using the automated Extract N Amp Plant PCR procedure on the Biomek FX Amplification of the 438 bp fragment of universal
5. chloroplast genomic DNA is indicated by the arrow M PCR marker Maize genomic DNA control No DNA template control PCR Analysis of Different Plant Types Maize Soybean M i HM EE ke M Universal Chloroplast Tobacco i Tomato Universal Chloroplast Figure 2 DNA was extracted from maize soybean tobacco and tomato leaves using the automated Extract N Amp Plant PCR procedure on the Biomek FX Amplification of the 400 500 bp fragment of universal chloroplast genomic DNA is indicated by the arrow M PCR marker Maize genomic DNA control No DNA template control Page 12 of 15 Cross contamination Analysis M 1 2 3 45 6 7 8 910 1112M 123 4 5 6 7 8 9 10 11 12 jy 4 Universal Chloroplast Universal Chloroplast 4 Universal Chloroplast Universal Chloroplast Figure 3 Tomato leaf disks 0 5 0 7 cm were placed in alternating wells of a 96 well plate The plate was then processed using the automated Extract N Amp Plant PCR procedure on the Biomek FX All samples were then subjected to amplification and 6 ul of the resultant products were electrophoresed on a 2 Agarose gel PCR products were not detected in the wells without plant tissue samples Quantitative PCR Analysis 1041 104 1 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle Figure 4 Eighty eight tomato leaf samples were extracted using the SYBR Green Extract N Amp Plant PCR Kit following the automate
6. extracts with a sealing film Plant tissue extracts can be stored for up to 6 months at 2 8 C B Methods 1 Page 6 of 15 Extract N Amp_Plant_PCRSetup Performs all of the steps necessary to extract DNA from 96 plant tissue samples and sets up the PCR reactions for the following kits XNAR XNAPR and XNAPRG The 96 channel head is used to prepare extracts and the Span 8 pod is used to prepare the PCR reactions from extracts and control DNA samples To perform PCR reaction setup there is a step in the method that calls up the PCR_Setup with controls method PCR_ Setup with controls Performs PCR reaction setup for 88 plant tissue samples and 8 controls using a master mix and transfers tissue DNA extracts using the Span 8 pod This method may be used independently of the Extract N Amp_Plant_PCRSetup described above if it is desired to perform additional amplification experiments from the tissue extracts PCR_Setup no controls Performs PCR reaction setup for 96 samples using a master mix and transfers tissue DNA extracts The Span 8 pod is used to transfer the master mix to the PCR plate and the 96 channel head is used to transfer extracts to the PCR plate This method may be used if it is desired to perform amplification experiments from the whole plate of tissue extracts without preparing PCR controls This method can also be called up in the Extract N Amp_Plant_PCRSetup method if it is desired to transfer extracts with the 9
7. plant leaves in a 96 well format The Extract N Amp Plant PCR Kits provide a novel extraction system that eliminates the need for long enzymatic digestions and homogenization steps that are not amenable to automation The XNAR Kit includes a specially formulated Extract N Amp PCR ReadyMix reagent that is a 2x reaction mixture of buffer salts dNTPs and Taq polymerase The ReadyMix reagent also contains Sigma s antibody mediated hot start mechanism JumpStart Taq polymerase for highly specific amplification of genomic DNA directly from the extract The XNAPR Kit includes the REDExtract N Amp PCR ReadyMix reagent containing an inert tracking dye for convenient direct loading of PCR reactions onto agarose gels for analysis The XNAPRG Kit includes a SYBR Green Extract N Amp PCR ReadyMix reagent for real time quantitative analysis of the amplified PCR products The validated method created for use on the Biomek FX Liquid Handling Workstation provides a walk away protocol for all aspects of the Extract N Amp Plant PCR Kits Extraction and amplification of genomic DNA from plant leaves is accomplished in 4 easy steps 1 The Extraction Solution is added to a piece of leaf tissue 2 Extracts are incubated for 10 minutes at 85 C 3 The Dilution Solution is added to the extract Extracts are now stable for at least 6 months if stored at 2 8 C 4 PCR reactions are set up using 4 ul of the extracts In just 20 minutes the Biomek FX can c
8. 3050 Spruce Street Saint Louis Missouri 63103 USA Telephone 800 325 5832 314 771 5765 SIGMA tn Productinformation Automated Protocol for Extract N Amp Plant PCR Kits Using the Biomek FX Workstation Beckman Coulter Extract N Amp Plant Product Codes XNAR XNAPR and XNAPRG Automation Guide 2 Description 2 ll Product Components 3 lll Storage 3 IV Materials to Be Supplied by the User 3 V Instrument Requirements for the Biomek FX Workstation 4 VI Temperature Control Device Watlow Setup 4 VII Plant Tissue Preparation 5 Vill Reagent Preparation 5 IX Automated Method Description 6 A Getting Started 6 B Methods 6 C Description of the Extract N Amp_Plant_PCRSetup Method T D Description of PCR_Setup with controls Method 8 E Description of PCR_Setup no controls Method 9 X Recommended Parameters for PCR Amplification 10 XI Method Customization 10 A Performing extraction without subsequent amplification 10 B Preparing 96 plant tissue extracts for PCR 11 C PCR setup only 11 D Use of a different PCR plate 11 E PCR setup using multiple primer sets 11 XII Performance Characteristics 12 XIII Troubleshooting 14 XIV Contact Information 15 Page 1 of 15 Automation Guide Il Description The Extract N Amp Plant PCR Kits Product Codes XNAR XNAPR and XNAPRG have been developed for use as a high throughput system for the rapid extraction and subsequent amplification of genomic DNA from various
9. 6 channel head C Description of the Extract N Amp_Plant_PCRSetup Method 1 Deck Layout Deck Position Equipment xtractSol L xtractior PCRPlate Swap W Eei a CRMaste P10 TRI LE Pelti Ew TL1 AP96 P250 Barrier Tips Sterile P2 AP96 P250 Barrier Tips Sterile P3 Swap P4 96 well reservoir for the Extraction Solution P6 Lid P7 96 well PCR plate with full skirt containing plant samples P8 96 well reservoir for Dilution Solution P12 142 column reservoir for PCR master mix ss P14 Span 8 P250 Barrier Tips P15 Span 8 P20 Barrier Tips ss i i lt lt C P46 24 position Eppendorf IsoTherm system DNA Control 96 well PCR plate with half skirt for PCR reaction setup Pa seated into a plate holder 2 Method Overview Below is a summary of the automated method Extract N Amp_Plant_PCRSetup For complete program details the automation program can be downloaded at www sigmaaldrich com automation 1 NOaRwWN Page 7 of 15 The extraction solution 50 pl is dispensed into a multiwell plate containing plant tissue samples using the 96 channel head The plate is moved to the shaker and mixed for 30 seconds The plate is moved to the Peltier ALP and heated for 10 minutes at 85 C The dilution solution 50 ul is dispensed into the plate containing the extracts Using the 96 channel head samples
10. VII Plant Tissue Preparation 1 Rinse a paper punch and forceps in 70 ethanol prior to use and between different samples Punch a 0 5 0 7 cm leaf tissue disk into a 96 well fully skirted PCR plate ensuring that each sample is centered down into the bottom of each well Chill the plate at 2 8 C until needed or flash freeze the samples on dry ice ethanol and keep at 70 C Vill Reagent Preparation 1 Extraction Solution To process a single plate of 96 samples add 15 ml of extraction solution to the 96 well reservoir S830018 located at P4 If it is desired to process more than 12 plates of samples the high profile reservoir S30014 is required Dilution Solution To process a single plate of 96 samples add 15 ml of dilution solution to the 96 well reservoir S30018 located at P8 If it is desired to process more than 12 plates of samples the high profile reservoir S30014 is required PCR Master Mix All Extract N Amp Plant PCR ReadyMixes are formulated as a 2x reaction mixture containing buffer salts dNTPs and Taq polymerase To prepare a PCR Master mix add water and the forward and reverse primers to the Extract N Amp Plant PCR ReadyMixes as described in the table below PCR ReadyMix j Stock Water E3004 R4775 or n fs irr a 4320 eee aa ii a PCR Master Mix 0 9 mi 1 5 ml 12 ul 12 ul 2 4 ml To set up 20 ul PCR reactions in one 96 well plate a total of 2 4 ml of PCR master mix needs to be added to the f
11. are mixed for 8 cycles The plate is moved to the shaker and mixed for 30 seconds A command calls up and performs all steps of the PCR_Setup with controls Method See below for explanation of the method D Description of PCR_Setup with controls Method xtractPle EZ Page 8 of 15 1 Deck Layout Deck Position Equipment P 96 well PCR plate with tissue DNA Extracts P414 96 well PCR amplification plate seated into a plate holder P12 12 column Reservoir for PCR master mix P14 Span 8 P250 Barrier Tips P15 Span 8 P20 Barrier Tips P16 24 position Eppendorf IsoThem system DNA Control CRMaste Pelti NAContra ED 2 Method Overview Below is a summary of the PCR Setup method using Span 8 to transfer 4 ul of DNA extracts For complete program details the automation program can be downloaded from www sigmaaldrich com automation 1 2 3 Wash the Span 8 dispense head with 2 ml of system fluid PCR master mix 16 ul is multi dispensed to PCR amplification plate using the Span 8 dispense head Tissue extract 4 ul is dispensed into the PCR amplification plate Control DNA samples 4 ul are dispensed to wells of A12 C12 E12 G12 of the PCR amplification plate using the Span 8 dispense head with tips 5 6 7 and 8 Water negative control 4 ul is dispensed to wells of B12 D12 F12 H12 of the PCR amplification plate using the Span 8 dispense head with tips 5 6 7 and 8 E Descript
12. d procedures Reaction analyses were performed on an ABI Prism 7700 Sequence Detection System The graph was plotted as the intensity of florescence in logarithms versus the value of cycle threshold Cr Page 13 of 15 XIII Troubleshooting Problem Little or no PCR product is detected Negative control shows a PCR product or false positive results are obtained Page 14 of 15 Cause A PCR component is missing or degraded No leaf tissue extract is added to the PCR reactions PCR reaction is inhibited due to contaminants in leaf tissue extract The mixing of Dilution Solution with leaf tissue DNA extract is not sufficient due to inefficient mixing by the Liquid Handler and or the clogging of the pipette tip by the tissue samples Genomic DNA is sheared when mix the solution with the pipettor Too few amplification cycles are performed Others Reagents are contaminated Solution Run a positive control to ensure components are functioning Check the performance of liquid handler Prime the system if needed Adjust the aspiration distance of the pipettors in the extraction plate Use less extract or dilute the extract with 50 50 mix of Extraction and Dilution Solutions and repeat PCR Increase the aspiration and dispensing speed and or cycle times in the mixing steps Increase the aspiration distance of the pipette tips in the mixing steps to avoid sucking up the tissue by the pipettors
13. ion of PCR_Setup no controls Method 1 Deck Layout EZ p W D C Deck Position Equipment P3 AP96 P20 Barrier Tips Sterile P7 96 well PCR plate with tissue DNA Extracts P11 96 well PCR amplification plate seated into a plate holder P12 12 column Reservoir for PCR master mix P14 Span 8 P250 Barrier Tips 2 Method Overview Below is a summary of the PCR Setup method using 96 channel head to transfer 4 ul of DNA extracts For complete program details download automation program from www sigmaaldrich com automation 1 Wash the Span 8 dispense head with 2 ml of system fluid 2 PCR master mix 16 ul is multi dispensed to PCR amplification plate using the Span 8 dispense head 3 Tissue extract 4 ul is dispensed into the PCR amplification plate using 96 channel head Page 9 of 15 X Recommended Parameters for PCR Amplification Step Temperature Time Cycles Extension 72 C 1 2 minutes 1 kb min Final Extension 72 C 10 minutes 1 Hold 4 C Indefinitely XI Method Customization A Performing extraction without subsequent amplification Tissue samples may be subjected to extraction without subsequent amplification To account for this modification step 7 in the Method Overview Section of Extract N Amp_Plant_PCRSetup method should be deleted and the deck layout in the Instrument Setup step needs to be updated as described in Section XI B Deck Position Equipment
14. irst column of the 12 column low profile reservoir 530028 located at P12 If setting up more than 3 plates of samples for PCR it will be necessary to use the high profile reservoir S30019 No template Control optional Add water into four 2 ml screw cap tubes and place in column 2 of the 24 position tube rack located at P16 DNA Controls optional Prepare genomic DNA controls for quantification of the plant tissue DNA extracts Genomic DNA from leaf tissues were prepared using GenElute Plant Genomic DNA Miniprep Kit and placed in column 1 of 24 position tube rack Page 5 of 15 IX Automated Method Description This overview describes the general liquid handling steps required to execute the automated Extract N Amp Plant PCR method and can be customized to a variety of applications For custom applications see Section XI A Getting Started 1 2 ae Turn on temperature control device Set up the deck layout by placing the tip boxes plates tube racks and reservoirs at the appropriate positions on the deck as described in Deck Layout Section Section IX C 1 IX D 1 or IX E 1 Add reagents to the appropriate reservoirs as described in Section VIII Run the method using Biomek Software Version 3 1 At the completion of the method place cap strips onto the PCR plate vortex to mix the solution and briefly centrifuge The PCR plate is now ready to be placed into a thermal cycler Seal the PCR plate containing plant tissue
15. omplete extraction and PCR reaction setup for 96 leaf tissue samples Page 2 of 15 ll Product Components Reagents Provided Extraction Solution Dilution Solution Extract N Amp PCR Ready Mix or SYBR Green Extract N Amp PCR Ready Mix Ill Storage Product Code Package Size E7526 D5688 E3004 for XNAR R4775 for XNAPR 4320 for XNAPRG Extract N Amp Plant XNAR 1 000 extractions 1 000 amplifications 120 ml 120 ml 12 ml REDExtract N Amp Plant XNAPR 1 000 extractions 1 000 amplifications 120 ml 120 ml 12 ml SYBR Green Extract N Amp Plant XNAPRG 1 000 extractions 1 000 amplifications 120 ml 120 ml 12 ml The Extract N Amp Plant PCR Kits can be stored at 2 8 C for up to 3 weeks For long term storage store at 20 C Do not store in a frost free freezer IV Materials to Be Supplied by the User IRN Plant leaf tissues Paper punch standard one hole Forceps small to medium in size Primers for plant genes of interest Optional GenElute Plant Genomic DNA Miniprep Kit Sigma G2N10 G2N70 and G2N250 for use as genomic DNA control 6 Water molecular biology reagent Sigma W4502 7 96 well PCR plates with full skirt Sigma P4616 8 96 well PCR plates with half skirt ABgene AB 1100 9 Lid universal Fisher 07200694 10 Ultra clear cap strip ABgene AB 0866 11 Corning plate holder Corning 6525 12 Sealing film SealPlate Sigma 2369659
16. the first run verify the performance of the Peltier ALP Manually set the temperature control device to the setting of 110 C with an offset of 4 C refer to the Watlow Temperature Control device User s Manual Place a PCR plate containing 100 ul of water in each well on the Peltier ALP and measure the temperature inside the wells using thermometer probes Verify that the temperature in the wells is at a minimum of 85 C after 3 minutes If well temperature does not reach a minimum of 85 C it will be necessary to adjust the offset Refer to User s Manual for directions on adjusting the offset Approximately one hour prior to running the automated method manually turn on the temperature control device and verify that the temperature display on the controller has reached the desired reading Using the Biomek software set both the Initialize and End Run Temperature settings to 110 C by selecting the Configuration Options for the Peltier ALP from the Device Editor menu as shown below Communications Port 4 al V Enable Temperature Control End Run Temperature fi 10 0 degrees C Initialize Temperature 110 0 degrees C 4t the beginning of the run or when the Initialize command is executed the temperature will be set to the value which was entered For Initialize Temperature t the end of each run the temperature will be set to the value which was entered For End Run Temperature Cancel Page 4 of 15
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