Automated Protocol for Extract-N-Amp™ Tissue - Sigma
Contents
1. Figure 3 DNA was extracted from mouse liver kidney pancreas and tails using the automated Extract N Amp Tissue PCR procedure on the Biomek FX workstation Amplification of the 1181 bp fragment of the IL 18 gene followed using 4 ul of extracted template DNA or 4 ul of human genomic DNA controls in a 20 ul PCR reaction incorporating the 2x PCR ReadyMix 6 ul of each reaction was loaded on a 1 agarose gel Page 15 of 17 XIII Troubleshooting Problem Little or no PCR product is detected Negative control shows a PCR product or false positive results are obtained Page 16 of 17 Cause A PCR component is missing or degraded No tissue extract is added to the PCR reactions PCR reaction is inhibited due to contaminants in the tissue extract PCR reaction is inhibited due to the presence of a precipitate that may form in the tissue extract The mixing of Neutralization Solution with 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 Genomic DNA is sheared when the solution is mixed with the pipettor Too few 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 dilute2. PCR setup only 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 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 PCR setup using multiple primer sets To amplify genomic DNA from the tissue extract with different primer sets primers can be added to microcentrifuge tubes and placed on the 24 position tube racks or added to the PCR ReadyMix and placed on different columns of 12 column reservoir 30028 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 Transfer of tissue extracts to a new plate Page 13 of 17 For long term storage of tissue extracts it is desirable to transfer them to a new plate To avoid clogging of the pipette tips with tissue samples it may be necessary to adjust the height of aspiration in step 10 described in the method overview for the Extract N Amp_Tissue_PCRSetup method In some instances manual transfer of the extracts to a new plate may be required When extracting DNA from tissue samples other than tail clips small pieces of tissue s
3. 3050 Spruce Street Saint Louis Missouri 63103 USA Telephone 800 325 5832 314 771 5765 Fax 314 286 7828 email techserv sial com sigma aldrich com Productinformation Automated Protocol for Extract N Amp Tissue PCR Kits Using the Biomek FX Beckman Coulter Extract N Amp Tissue Product Codes XNATR and XNAT2R 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 Tissue Preparation 4 Vil Reagent Preparation 5 VIII Temperature Control Device Watlow Setup 6 IX Automated Method Description 6 A Getting Started 6 B Biomek Methods T C Description of the Extract N Amp_Tissue_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 11 Performing extraction without subsequent amplification 11 Preparing 96 tissue extracts for PCR 12 PCR setup only 13 Use of a different PCR plate 13 PCR setup using multiple primer sets 13 Transfer of tissue extracts to a new plate 13 XII Performance Characteristics 14 XIII Troubleshooting 16 XIV Contact Information 17 Page 1 of 17 Automation Guide Il Description The Extract N Amp Tissue PCR Kits XNATR and XNAT2R have been developed for use as a high throughput system for the rapid extraction a
4. amples may float in the prepared extracts To avoid clogging of the pipette tips with tissue samples it may be required to centrifuge down the extracts in the Extraction plate prior to transfer to a new plate XII Performance Characteristics Automated Method for the Extract N Amp PCR Analysis of Mouse Tail Samples M 12 3 4 5 6 7 8 9 10 11 M 492 34 5 67 8 91011 M TEUER T AREE E deb ouch HD ou ey dt ty enias ne ea conan Gm ma eo IL 16 IL 16 oe IL 18 IL 16 Figure 1 DNA was extracted from 88 samples of mouse tails 0 3 0 4 cm using the automated Extract N Amp Tissue PCR procedure on the Biomek FX workstation Amplification of the 1181 bp of the IL 18 gene followed using 4 pl of extracted template or 4 ul of human genomic DNA controls in a 20 ul PCR reaction incorporating the 2x PCR ReadyMix 6 ul of each reaction was analyzed on a 1 Agarose gel Page 14 of 17 Cross Contamination Analysis 123 45 6 7 8 9 1011124 12 3 4 5 6 7 8 9 101112 m lt lt IL 1f8 Figure 2 Mouse tails were placed in alternating wells of the extraction plate The extraction plate was processed using the automated Extract N Amp Tissue PCR procedure on the Biomek FX workstation All samples were amplified and 6 ul of the resultant products were electrophoresed on a 1 agarose gel No PCR products were detected in the wells without tissue samples Multiple Tissue Samples Liver Kidney IL 16 Pancreas Tail MES O teM SM IL 16
5. e extracts for 30 seconds at 750 rpm Pause Shaker for a 10 minute incubation at room temperature Gripper tool is used to move plate containing tissue extracts from shaker to Peltier ALP Pause Peltier ALP for a 15 minute incubation at 85 C The lid is removed from the plate containing tissue extracts and placed at P6 Gripper tool is used to move plate containing tissue extracts from Peltier ALP to P7 50 ul of Neutralization Solution is aspirated from a reservoir and dispensed into the multiwell plate with the tissue extracts by the 96 channel head The 96 channel head is used to pipette mix the extracts for 8 cycles and then transfer 80 ul of tissue extract to a new plate for the storage 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 Page 8 of 17 1 Deck Layout P10 ransferP CRPlate Hi Pelti Deck Position Equipment CRMaste E ntro P11 96 well PCR plate with tissue DNA Extracts P12 96 well PCR amplification plate seated into a plate holder P13 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 2 Method Overview Below is a summary of the PCR Setup method using Span 8
6. es 96 well reservoir for Neutralization Solution P8 P11 96 well PCR plate for transferring neutralized tissue extracts for long term storage Page 11 of 17 Preparing 96 tissue extracts for PCR It may be desired to extract DNA from 96 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_Tissue_PCRSetup method 1 Click on the Run PCR_Setup with controls step of the Extract N Amp Tissue_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 Tissue_PCRSetup and PCR_Setup no controls methods as following O E3 xtractior kea p xtraction cee CRPlate eutraSoln xtraction CRMaste Deck Position Equipment TL1 AP96 P250 Barrier Tips Sterile P2 AP96 P250 Barrier Tips Sterile AP96 P20 Barrier Tips Sterile P3 P4 96 well reservoir for the mixture of Extraction and Tissue Preparation Solution P5 Swap P6 Lid P7 96 well PCR plate with full skirt containing tissue samples 96 well reservoir for Neutralization Solution P8 P11 96 well PCR plate for transferring neutralized tissue extracts for long term storage P12 96 well PCR amplification plate seated into a plate holder P13 12 column reservoir for PCR Master Mix P14 Span 8 P250 Barrier Tips Page 12 of 17
7. hs 4 PCR reactions are set up using 4 ul of the extracts In just 35 minutes the Biomek FX can complete DNA extraction of and PCR reaction setup for 96 tissue samples Page 2 of 17 ll Product Components Reagents Provided Product Code Extract N Amp Tissue REDExtract N Amp Tissue XNAT2R XNATR p 1 000 extractions 1 000 extractions Package Size 1 000 amplifications 1 000 amplifications Extraction Solution E7526 240 ml 240 ml Tissue Preparation T3073 30 ml 30 ml Solution Neutralization Solution B N3910 240 ml 240 ml Extract N Amp PCR Ready Mix or REDExtract 3004 for XNAT2R 42ml 12 mi N Amp PCR Ready Mix R4775 for XNATR Ill Storage The Extract N Amp Tissue 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 CMNOANRWN gt Animal Tissues Small dissecting scissors Forceps small to medium in size Primers for genes of interest Molecular biology grade water Sigma W4502 96 well PCR plates with full skirt Sigma P4616 96 well PCR plates with half skirt ABgene AB 1100 Lid universal Fisher 07200694 Ultra clear cap strip ABgene AB 0866 Corning plate holder Corning 6525 Sealing film SealPlate Sigma Z369659 Microcentrifuge tubes 1 5 ml 2 ml screw cap 24 position Eppendorf lsoTherm System Fisher 05 405 22 12 column reagent reservoir with low pr
8. late ensuring that each sample is centered down into the bottom of each well 2 Chill the plate at 2 8 C until needed Other Animal Tissues 1 Rinse scissors and forceps in 70 ethanol prior to use and between different samples Place a 4 6 mg piece of tissue into a 96 well PCR plate ensuring that each sample is centered down into the bottom of each well 2 Chill the plate at 2 8 C until needed Page 4 of 17 Vil Reagent Preparation 1 Extraction and Tissue Preparation Solution Mixture Pre mix the Extraction and Tissue Preparation Solutions at a ratio of 4 1 This mixture can be stored for up to 2 hours before use To process a single plate of 96 samples add 18 ml of the mixture to the 96 well reservoir located at position P4 see Section IX for deck layout If it is desired to process more than 12 plates of samples the high profile reservoir S30014 is required 2 Neutralization Solution To process a single plate of 96 samples add 20 ml of Neutralization Solution to the 96 well reservoir located at position P8 see Section IX for deck layout If it is desired to process more than 12 plates of samples the high profile reservoir S30014 is required 3 PCR Master Mix The Extract N Amp Tissue PCR ReadyMix is a 2X reaction mixture containing buffer salts dNTPs and Taq polymerase To prepare a PCR Master Mix add water and primers forward and reverse to the Extract N Amp Tissue PCR ReadyMix as described in table bel
9. nd subsequent amplification of genomic DNA in a 96 well format from mouse tails and other animal tissues The Extract N Amp Tissue PCR Kits provide a novel DNA extraction system eliminating the need for long enzymatic digestions and homogenization steps that are not amenable to automation The XNAT2R Kit includes a specially formulated Extract N Amp PCR ReadyMix reagent that includes a 2X reaction mixture of buffer salts dNTPs and Taq polymerase The ReadyMix reagent also contains Sigma s antibody mediated hot start polymerase JumpStart Taq polymerase for highly specific amplification of genomic DNA directly from the extract The XNATR Kit includes the REDExtract N Amp PCR ReadyMix that also contains an inert tracking dye for convenient direct loading of the PCR reactions onto an agarose gel for analysis The automated method created and validated for use on the Biomek FX Liquid Handling Workstation from Beckman Coulter provides a walk away protocol for all aspects of the Extract N Amp Tissue PCR Kits Extraction and amplification of genomic DNA from animal tissues is accomplished in 4 easy steps 1 The Extraction and Tissue Preparation Solution mixture is added to tissue samples and incubated at room temperature for 10 minutes 2 Extracts are incubated for 15 minutes at 85 C 3 A Neutralization Solution is added to the extract a Once the Neutralization Solution has been added extracts can be stored at 4 C for at least 6 mont
10. ng the automated method manually turn on the temperature control device and verify that the temperature display on the controller has reached the desired reading In Biomek software set both Initialize and End Run Temperature settings at 110 C by selecting the Configuration Options for the Peltier ALP from the Device Editor menu as shown below Communications Port 4 J Enable Temperature Control End Run Temperature fi 10 0 degrees C Initialize Temperature fi 10 0 degrees C t 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 IX Automated Method Description This overview describes the general liquid handling steps required to execute the automated Extract N Amp Tissue PCR method and can be customized to a variety of applications To customize applications see Section XI A Getting Started 1 2 3 4 5 Page 6 of 17 Turn on temperature control device Set up deck layout place the tip boxes plates tube rack and reservoirs at the appropriate positions on the deck as described in Deck Layout Section Add reagents to the appropriate reservoirs as described in Section VI Run the method using Biomek Software Version 3 1 At the completion of the method place cap st
11. ofile Innovative Microplates S30028 96 well reservoir with low profile and pyramidal bottom Innovative Microplates S30018 Optional High profile 12 column reagent reservoir Innovative Microplates S30019 Optional High profile 96 well reservoir with pyramidal bottom Innovative Microplates 830014 Thermal Cycler Thermometer Fisher 15 077 26 Page 3 of 17 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 2 PO w A A o Sm A A So om om VI Tissue Preparation For Fresh or Frozen Mouse Tails 1 Rinse scissors and forceps in 70 ethanol prior to use and between different samples Place a 0 3 0 4 cm piece of mouse tail tip cut end down into a 96 well PCR p
12. on plate using the Span 8 dispense head Steps 3 and 4 are repeated 3 more times until the Span 8 has dispensed 16 ul of PCR Master Mix to all 96 wells of the PCR amplification plate 200 ul barrier tips are removed from the Span 8 dispense head 4 ul of tissue extract is aspirated from the multiwell plate containing tissue extracts using 96 channel head Tissue extract is dispensed into the PCR amplification plate X Recommended Parameters for PCR Amplification Page 10 of 17 Step Temperature Time Cycles Initial Denaturation 94 96 C 3 minutes 1 Denaturation 94 96 C 0 5 1 minute Annealing 45 68 C 0 5 1 minute 30 40 Extension 72 C 1 2 minutes 1 kb min Final Extension 72 C 10 minutes 1 Hold 4 C Indefinitely XI Method Customization Performing extraction without subsequent amplification Tissue samples may be subjected to extraction without subsequent amplification To account for this modification step 11 in the Method Overview Section of Extract N Amp_Tissue_PCRSetup method should be deleted and the deck layout in the Instrument Setup step needs to be updated as following xtractSol eutraSoln DF xtractior ransferP Swap xtraction eutraliza lie HE Deck Position Equipment TL1 AP96 P250 Barrier Tips P2 AP96 P250 Barrier Tips P3 Swap P4 96 well reservoir for the mixture of Extraction and Tissue Preparation Solution P6 Lid P7 96 well PCR plate with full skirt containing tissue sampl
13. ow PCR ReadyMix Forward Primer Reverse Primer Stock Water E3004 100 uM 100 uM PCR Master Mix 0 9 ml 15 ml 12 ul 12 ul 2 4 ml To set up 20 ul PCR reactions in one 96 well plate add 2 4 ml of PCR Master Mix to the first column of the 12 column reservoir located at position P13 see Section IX for deck layout If setting up more than 3 plates of samples for PCR it will be necessary to use reservoir S30019 4 No template Control optional Add water to four 2 ml screw cap tubes and place in column 2 positions 5 8 of the 24 position tube rack 5 DNA Controls optional Prepare genomic DNA controls for quantification of tissue DNA extracts Prepare 4 tubes containing 10 ng ul 5 ng pl 1 25 ng ul and 0 31 ng ul samples of genomic DNA and place in column 1 positions 1 4 of the 24 position tube rack Page 5 of 17 Vill Temperature Control Device Watlow Setup Prior to the first run of the automated method 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 User s Manual Place a PCR plate 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 may be necessary to adjust the offset refer to User s Manual Approximately one hour prior to runni
14. rips 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 tissue extracts with a sealing film Tissue extracts can be stored for up to 6 months at 4 C B Biomek Methods 1 Extract N Amp_Tissue_PCRSetup Performs all of the steps necessary to extract DNA from 96 tissue samples and setup PCR reactions The 96 channel head is used to prepare extracts and the Span 8 is used to prepare 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 tissue samples and 8 controls using a Master Mix and transfers tissue DNA extracts using Span 8 This method may be used 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 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 Tissue_PCRSetup method if it is desired to transfer extracts with
15. the extract with 50 50 mix of Extraction and Neutralization Solutions and repeat PCR Centrifuge the plate containing tissue extracts before adding the extracts to PCR amplification plate Increase the aspiration and dispensing speed and or cycle times in the mixing steps Decrease the aspiration distance of the pipette tips in the mixing steps to avoid sucking up the tissue by the pipettors 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 at a time Refer to the Technical Bulletin of Extract N Amp Tissue 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 Page 17 of 17
16. the 96 channel head C Description of the Extract N Amp_Tissue_PCRSetup Method 1 xtractSol Pelti CRMaste NAContra Orbital1 P5 Deck Layout O E xtractior ransferP Swap Eei cee CRPlate Deck Position Equipment TL1 P2 AP96 P250 Barrier Tips Sterile AP96 P250 Barrier Tips Sterile P3 P4 P6 P7 P8 P11 P12 P13 P14 P15 P16 Page 7 of 17 Swap 96 well reservoir for the mixture of Extraction and Tissue Preparation Solution Lid 96 well PCR plate with full skirt containing tissue samples 96 well reservoir for Neutralization Solution 96 well PCR plate with full skirt for transferring neutralized tissue extracts 96 well PCR plate with half skirt for PCR reaction setup seated into a plate holder 12 column reservoir for PCR Master Mix Span 8 P250 Barrier Tips Span 8 P20 Barrier Tips 24 position Eppendorf IsoTherm system DNA Control 2 Method Overview Below is a summary of the Extract N Amp Tissue automated method For complete program details download automation program at www sigmaaldrich com automation 1 ak OND 10 11 62 5 ul of Extraction and Tissue Preparation Solution mixture is aspirated from a reservoir and dispensed into multiwell plate containing tissue samples by the 96 channel head Gripper tool is used to move the plate containing tissue extract to the shaker Shaker is activated to begin mixing plate with tissu
17. tion plate using the Span 8 dispense head with tips 5 6 7 and 8 Refresh 20 ul barrier disposable tips 4 ul of water are aspirated from four microcentrifuge tubes and 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 Refresh 20 pl barrier disposable tips E Description of PCR_Setup no controls Method Page 9 of 17 1 Deck Layout Orbital Deck Position Equipment P3 AP96 P20 Barrier Tips Sterile P11 96 well PCR plate with tissue DNA Extracts P412 96 well PCR amplification plate seated into a plate holder P13 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 2 3 Wash the Span 8 dispense head with 2 ml of system fluid 200 ul barrier disposable tips are loaded onto the Span 8 dispense head PCR Master Mix is aspirated from the 12 column reservoir using the Span 8 dispense head The Span 8 is acting like a bulk reagent dispenser and is aspirating enough reagents to dispense to a quarter of the plate 16 ul of PCR Master Mix is multi dispensed to PCR amplificati
18. to transfer 4 ul of DNA extracts For complete program details download automation program from www sigmaaldrich com automation 1 2 3 10 11 12 Wash the Span 8 dispense head with 2 ml of system fluid 200 ul barrier disposable tips are loaded onto the Span 8 dispense head PCR Master Mix is aspirated from the 12 column reservoir using the Span 8 dispense head The Span 8 is acting like a bulk reagent dispenser and is aspirating enough reagents to dispense to a quarter of the plate 16 ul of PCR master mix is multi dispensed to the PCR amplification plate using the Span 8 dispense head Steps 3 and 4 are repeated 3 more times until the Span 8 has dispensed 16 ul of PCR master mix to all 96 wells of the PCR amplification plate 200 ul barrier tips are removed from the Span 8 dispense head 4 ul of tissue extract is aspirated from the multiwell plate containing tissue extracts with Span 8 dispense head Tissue extract is dispensed into the PCR amplification plate Because the Span 8 dispense head can only perform operations eight wells at a time a loop is created to account for all samples Steps 7 10 are repeated 10 times or the number of times as needed New 20 ul barrier disposable tips are used for each transfer 20 ul barrier disposable tips are removed from the Span 8 dispense head 4 ul of control DNA samples are aspirated from four microcentrifuge tubes and dispensed to wells of A12 C12 E12 G12 of the PCR amplifica
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