Automated Protocol for Extract-N-Amp™ Tissue - Sigma
Contents
1. Page 5 of 11 Vil Temperature Control Device Setup Set the temperature control device to the maximum 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 nl of water in each well on the device 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 Approximately one hour prior to running the automated method turn on the temperature control device and verify that the temperature display on the controller has reached the desired reading VIII Tissue Preparation For Fresh or Frozen Mouse Tails 1 2 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 plate ensuring that each sample is centered down into the bottom of each well 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 Chill the plate at 2 8 C until needed Reagent Preparation Extraction and Tissue Preparation Solution Mixture Pre mix the Extraction and Tissue Preparation Solutions at a ratio of 4 1 This solution can be store2. Extract N Amp Tissue REDExtract N Amp Tissue XNAT2R XNATR Package Size 1000 extractions 1000 extractions 1000 amplifications 1000 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 E3004 for XNAT2R 12 ml 12 ml Ready Mix or REDExtract R4775 for XNATR N Amp PCR Ready Mix 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 use a frost free freezer IV CMNONRWN gt Materials to Be Supplied by the User Animal tissues Small dissecting scissors Forceps small to medium in size Primers for genes of interest Water molecular biology reagent Sigma W4502 96 well PCR plates with full skirt Sigma P4616 Lid universal Fisher 07200694 96 well PCR plates Stratagene 410088 Cap strips Stratagene 410096 PCR plate holder Nunc 251357 5 ml polypropylene round bottom tube 12 x 75 mm Microcentrifuge tubes 1 5 ml or 2 ml Aluminum sealing film Sigma A2350 Heating device for 96 well plate Inheco Industrial Heating amp Cooling e CPAC UltraFlat High Temperature 7000091 e TEC Control With RS 232 Interface 8900009 96 well PCR Plate Adapter 3200203 Thermal cycler RoboCycler Stratagene Thermometer Fisher 15 077 26 Page 3 of 11 V Instrument Requirements for the Freedom EVO 1
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 Tecan Freedom EVO 150 Workstation Extract N Amp Tissue Product Codes XNATR and XNAT2R Automation Guide l Description ll Product Components Ill Storage IV Materials to Be Supplied by the User V Instrument Requirements for the Freedom EVO 150 Workstation VI Worktable Setup Vil Temperature Control Device Setup VIII Tissue Preparation IX Reagent Preparation X Automated Method Description A Getting Started B Method Overview XI Recommended Parameters for PCR Amplification XII Method Customization A Use of a different PCR plate B PCR setup using multiple primer sets C Transfer of tissue extracts to a new plate Oo ooo DO NNN OOO aA A WW WD ND XIII Performance Characteristics XIV Troubleshooting XV Contact Information Page 1 of 11 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 and subsequent amplification of genomic DNA from mouse tails and other animal tissues in a 96 well format The Extract N Amp Tissue PCR Kits provide a novel DNA extraction system eliminating the need for long enzymatic digestions an
4. 50 Workstation For detailed ordering information contact Tecan sales representative Part Description Qty LiHa Arm 8 channel with Disposable Tip Option RoMa Arm 1 ml Syringes DiTi 3 Position DiTi 2 Position with Waste Slide and Cover Wash Station Te Shake Microplate Carrier Landscape 3 Position MP Hotel 9 Position 16 Position Tube Carrier 16 Position Microcentrifuge Tube Carrier 100 ml Trough Carrier 3 Position 100 ml Trough 25 ml Trough NNa Sa A S A 32 3 Page 4 of 11 VI Worktable Setup otel 9Pos Mic Grid Position Position Equipment DiTi 3 position Position 1 200 ul tips DiTi 2 position with waste slide and cover Position 1 1000 tl tips Position 2 10 ul tips Position 3 DiTi waste slide and cover 14 Wash Station Position 1 cleaner shallow Position 2 waste oe Position 1 100 ml trough with Neutralization Solution Position 3 100 ml trough with Extraction Mixture Te Shake with 96 well PCR Te Shake with 96 well PCR plate containing tissue samples containing tissue samples 13 mm 16 position tube rack Position 16 PCR Master Mix 13 mm 16 position tube rack Positions 9 16 Control samples 3 position Microplate Carrier Landscape Position 1 Lid Position 2 96 well PCR plate for the transfer of neutralized tissue extracts Position 3 PCR amplification plate 35 Heating Device 37 MP Hotel for temporary storage of lid at position 1
5. DS i l ed ee el ce IL 16 1181 bp cai bnd id d t et toe Oe ls ee M lM ee a ee oe et oe IL 16 1181 bp i IL 16 IL 16 e o e b es ae ee b ai et es OS l ey G ee oe es 1181 bp Figure1 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 Tecan Freedom EVO workstation Amplification of the 1181 bp IL 18 gene followed using 4 ul 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 were analyzed on a 1 Agarose gel M PCR marker Mouse genomic DNA control No DNA template control Cross Contamination Analysis M123 45 67 8 9101112 M123 4 5 6 78 9101112M IL 16 1181 bp D IL 16 ge 1181 bp IL 16 1181 bp 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 Tecan Freedom EVO 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 Page 9 of 11 XIV Troubleshooting Problem Little or no PCR product is detected Negative control shows a PCR product or false positive results are obtained Page 10 of 11 Cause A PCR component is missing or degraded No tissue extrac
6. Recommended Parameters for PCR Amplification Step Temperature Time Cycles Initial Denaturation 94 96 C 3 minutes 1 Denaturation 94 96 C 0 5 1 minutes Annealing 45 68 C 0 5 1 minutes 30 40 Extension 72 C 1 2 minutes 1 kb min Final Extension 72 C 10 minutes 1 Hold 4 C Indefinitely XII Method Customization A Use of a different PCR plate The automated method was created using the 96 well PCR amplification plates with half skirt from Stratagene Other PCR plates including 384 well plates may be used in this method but may require the creation of a new labware in the Freedom EVOware software B PCR setup using multiple primer sets To amplify genomic DNA from the 96 tissue extracts with different primer sets primers can be added to microcentrifuge tubes and placed on the tube racks or added to the PCR ReadyMix and placed into the additional 100 ml or 25 ml troughs on the appropriate carriers Additional steps will need to be added to the automated program C Transfer of tissue extracts to a new plate Page 8 of 11 Because the size of tissue samples may vary it may be necessary to adjust the height of aspiration in the method to avoid clogging of the pipet tips with tissue samples In some instances manual transfer of the extracts to a new plate may be required XIII Performance Characteristics PCR Analysis of Mouse Tails Samples M123 4 5 6 78 91011 M1234 5 6 78 9 1011 M ed ss ee ee et ee et et ey ee
7. d for up to 2 hours before use To process a single plate of 96 samples add 10 ml of the mixture to the 100 ml trough at grid location 16 position 3 Neutralization Solution To process a single plate of 96 samples add 10 ml of Neutralization Solution to the 100 ml trough at grid location 16 position 1 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 Master mix add water and primers forward and reverse to the Extract N Amp Tissue PCR ReadyMix as described in table below Water PCR Mix Forward Primer Reverse Primer Stock E3004 100 uM 100 uM Working 2 ml 0 73 ml 1 25 ml 10 ul 10 ul To set up 20 ul PCR reactions in one 96 well plate a total of 2 ml PCR master mix needs to be added to the 5 ml tube at grid location 25 position 16 Page 6 of 11 X 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 XII A Getting Started 1 2 a amp Turn on the temperature control device Set up the worktable by placing the carriers and racks at the appropriate grid positions as described in section VI Add reagents to the appropriate troughs as described in section IX Run the method using Freedom EVOware Software Version 1 0 SP1 or later At th
8. d homogenization steps that are not amenable to automation The XNAT2R 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 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 reagent containing an inert tracking dye for convenient direct loading of the PCR reactions onto an agarose gel for analysis The validated method created for use on the Freedom EVO 150 Liquid Handling Workstation from Tecan provides a high throughput protocol for all aspects of the Extract N Amp Tissue PCR kit 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 Once the Neutralization Solution has been added extracts can be stored at 2 8 C for at least 6 months 4 PCR reactions are set up using 4 ul of the extracts In just 50 minutes the Freedom EVO 150 can complete the extraction and PCR reaction setup for 96 tissue samples Page 2 of 11 ll Product Components Reagents Provided Product Code
9. e 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 tissue extracts with a sealing film Tissue extracts can be stored for up to 6 months at 2 8 C B Method Overview The ExtractNAmpTissue method performs all of the steps necessary to extract DNA from 96 tissue samples and set up PCR reactions for 96 samples using a master mix For complete program details download the automation program at www sigmaaldrich com automation Page 7 of 11 1 Set DiTi positions for 1000 ul 200 ul and 10 ul disposable tips Extraction and Tissue Preparation Solution mixture 62 5 ul is dispensed into each well of the Extraction plate containing the tissue samples Mix the Extraction plate by shaking at 750 rpm for 30 sec Pause for a 10 minute incubation Transfer the Extraction plate to the heating device for an incubation of 15 minutes at 85 C 6 Neutralization solution 50 ul is dispensed into each well of the Extraction plate 7 Neutralized tissue extracts 80 ul are transferred from the Extraction plate to the Transfer plate for long term storage 8 PCR master mix 16 ul is dispensed into each well of the PCR plate 9 Tissue extracts 4 ul are dispensed into each well of the PCR plate 10 DNA controls 4 ul are dispensed into wells of column 12 of the PCR plate oO amp XI
10. er to the Technical Bulletin of Extract N Amp Tissue PC 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 XV 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 Freedom EVO and Freedom EVOware are registered trademarks of Tecan Trading AG 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 slip
11. t 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 pipet 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 position of the disposable tips in the extraction plate if the liquid detection function is inactivated Use less extract or dilute 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 pipet 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 Ref
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