Automated Protocol for Extract-N-Amp™ Plant PCR - Sigma
Contents
1. Please see reverse side of the invoice or packing slip2. the plant tissue samples Mix the Extraction plate by shaking at 750 rpm for 30 seconds Transfer the Extraction plate to a heater for an incubation of 10 minutes at 85 C Dilution solution 50 pl is dispensed into each well of the Extraction plate PCR master mix 16 ul is dispensed into all the wells of the PCR plate Plant tissue extracts 4 ul are dispensed into each well of the PCR plate DNA controls 4 ul are dispensed into wells of column 12 of the PCR plate N ONAARW XI Recommended Parameters for PCR Amplification Step Temperature Initial Denaturation 94 96 C Denaturation 94 96 C Annealing 45 68 C Extension 72 C Final Extension 72 C Hold 4 C XII Method Customization A Use of a different PCR plate Time 3 minutes 0 5 1 minutes 0 5 1 minutes 1 2 minutes 1 kb min 10 minutes Indefinitely Cycles 30 40 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 Page 8 of 12 To amplify genomic DNA from the 96 plant 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 ca
3. E3004 for XNAR 12 ml 12 ml Ready Mix R4775 for XNAPR Ill Storage 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 use a frost free freezer IV Materials to Be Supplied by the User IBON Plant leaf tissues Paper punch standard one hole Forceps small to medium in size Primers for genes of interest Optional GenElute Plant Genomic DNA Miniprep Kit Sigma G2N10 for use as genomic DNA control 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 e 96 well PCR Plate Adapter 3200203 Thermal cycler Thermometer Fisher 15 077 26 Page 3 of 12 V Instrument Requirements for the Freedom EVO 150 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 Sta
4. wells of a 96 well plate The plate was then processed using the automated Extract N Amp Plant PCR procedure on the Tecan Freedom EVO workstation 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 Page 10 of 12 XIV Troubleshooting Problem Little or no PCR product is detected Negative control shows a PCR product or false positive results are obtained Page 11 of 12 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 the solution is mixed 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 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 Dilution Solutions and repeat PCR Incre
5. 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 Tecan Freedom EVO 150 Workstation Extract N Amp Plant Product Codes XNAR and XNAPR 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 Vill Plant 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 XIII Performance Characteristics 0O DO NNN OOO aA BP WW WD ND XIV Troubleshooting N XV Contact Information Page 1 of 12 Automation Guide Il Description The Extract N Amp Plant PCR Kits XNAR and XNAPR have been developed for use as a high throughput system for the rapid extraction and subsequent amplification of genomic DNA from various 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 Read
6. ase the aspiration and dispensing speed and or cycle times in the mixing steps Raise the aspiration position 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 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 the 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
7. e 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 Plant Tissue Preparation l 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 Reagent Preparation Extraction Solution To process a single plate of 96 samples add 10 ml of Extraction Solution to the 100 ml trough at grid location 16 position 3 Dilution Solution To process a single plate of 96 samples add 10 ml of Dilution Solution to the 100 ml trough at grid location 16 position 1 PCR Master Mix The Extract N Amp Plant 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 Plant PCR ReadyMix as described in table below Water PCR Mix Forward Primer Reverse Primer Stock E3004 100 uM 100 uM Working 2 ml 0 75 ml 1 25 ml 3 5 ul 3 5 ul To set up 20 ul PCR reactions in one 96 well plate a to
8. rriers Additional steps will need to be added to the automated program XIII Performance Characteristics PCR Analysis of Tobacco Leaf Tissue Samples M123 4567 8 91011 M123 45 67 8 91011 M B se es es i iMi M lt lt Universal Chloroplast D lt Universal Chloroplast F PPOP tee tts tna eh ae E de o Universal Chloroplast H Universal Chloroplast Figure 1 DNA was extracted from 88 Tobacco leaf samples The 96 well plate was processed using the automated Extract N Amp Plant PCR procedure on the Tecan Freedom EVO workstation Amplification of the 438 bp fragment of universal 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 Tobacco Tomato lt q 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 Tecan Freedom EVO workstation Amplification of the 438 bp fragment of universal chloroplast genomic DNA is indicated by the arrow M PCR marker Maize genomic DNA control No DNA template control Page 9 of 12 Cross Contamination Analysis M 12345678 9 101112M 12 34 567 8 9101112M A Universal Chloroplast c Universal Chloroplast E Universal Chloroplast G H Universal Chloroplast Figure 3 Tobacco leaf disks 0 5 0 7 cm were placed in alternating
9. tal of 2 ml PCR master mix needs to be added to the 5 ml tube at grid location 25 position 16 Page 6 of 12 X 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 To customize applications see Section XIl A Getting Started 1 2 ak w Turn on 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 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 extracts with a sealing film Plant tissue extracts can be stored for up to 6 months at 2 8 C B Method Overview Page 7 of 12 The ExtractNAmpPlant method performs all of the steps necessary to extract DNA from 96 plant tissue samples and set up PCR reactions using a master mix For complete program details download the automation program at www siqmaaldrich com automation Set DiTi positions for 1000 ul 200 ul and 10 ul disposable tips Extraction solution 50 ul is dispensed to each well of the Extraction plate containing
10. tion Te Shake Microplate Carrier Landscape 3 Position MP Hotel 9 Position 16 Position Tube Carrier 16 Position Eppendorf Tube Carrier 100 ml Trough Carrier 3 Position 100 ml Trough 25 ml Trough NNa A Am Sa So S 3 OE Page 4 of 12 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 ul tips Position 2 10 ul tips Position 3 DiTi waste slide and cover 14 Wash Station Position 1 cleaner shallow Position 2 waste Position 1 100 ml trough with Dilution Solution Position 3 100 ml SSSI wines POS a I ae with Extraction Solution Te Shake with 96 well PCR Te Shake with 96 well PCR plate containing plant tissue samples containing plant 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 3 PCR amplification plate 35 Heating Device 37 MP Hotel for temporary storage of lid at position 1 Page 5 of 12 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 ul of water in each well on the device and measure the temperatur
11. yMix 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 the PCR reactions onto agarose gels 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 Plant PCR kit 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 35 minutes the Freedom EVO 150 can complete the extraction and PCR reaction setup for 96 plant tissue samples Page 2 of 12 ll Product Components Reagents Provided Product Code Extract N Amp Plant REDExtract N Amp Plant XNAR XNAPR Package Size 1000 extractions 1000 extractions 1000 amplifications 1000 amplifications Extraction Solution E7526 120 ml 120 ml Dilution Solution D5688 120 ml 120 ml Extract N Amp PCR
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