E-Z 96 Plant DNA DS Kit - Omega Bio-Tek
Contents
1. Seal the 96 well Racked Microtubes with Caps for Racked Microtubes Store DNA at 20 C 7 96 Plant DNA DS Kit Protocols E Z 96 Plant DNA DS Kit Vacuum Protocol The following protocol is based on using Omega Bio tek s vacuum manifold VAC 03 Materials and Equipment to be Supplied by User e Vacuum manifold and vacuum source e Centrifuge equipped with swing bucket rotor capable of at least 3 000 x g Water bath oven or incubator capable of 80 C Vortexer 10096 ethanol Liquid nitrogen for freezing disrupting samples For Fresh Frozen Specimens Equipment for disrupting plant tissue Ice freezer or 96 well cryorack at 20 C Sealing film Before Starting Prepare HBC Buffer and DNA Wash Buffer according to Preparing Reagents section on Page 5 Set a water bath oven or incubator to 65 C Heat Elution Buffer to 65 C 1 Transfer up to 10 mg dry powdered tissue or 50 mg fresh or frozen tissue to 96 well Round well Plate provided and seal with Caps for Round well Plate Note No more than 50 mg wet weight or 10 mg dry weight starting material is recommended More or less can be used depending on results Water content and buffer absorption of samples affect optimal starting amounts 2 plant tissue following one of the methods described in the Disruption of Plant Tissue section on Pages 8 If homogenizing in the presence of lysis buffer with fres2. 2 96 Plant DNA DS Kit D1411 01 1 x 96 preps D1411 02 4 x 96 preps August 2015 2 96 Plant DNA DS Kit Table of Contents OTT 2 Illustrated ProLto6ols insano 3 Kit Contents Storage and Stability 4 Preparing Reagents Cleaning 5 Guidelines for Vacuum Manifold sss 6 Disruption of Plant Tis ui iria 8 E Z 96 Plant DNA DS Centrifugation Protocol 10 E Z 96 Plant DNA DS Vacuum Protocol 14 Troubleshooting Guide esencias 19 Order sica 20 Manual Revision August 2015 5 OMEGA bio tek Innovations in nucleic acid isolation Introduction The E Z 96 Plant DNA DS Mini Kit is designed for efficient recovery of genomic DNA up to 30 kb in size from fresh frozen or dried plant tissue samples rich in polysaccharides polyphenols or those having a lower DNA content Up to 50 mg wet tissue can be processed in less than 1 hour The system combines the reversible nucleic acid binding properties of the HiBind matrix with the speed and versatility of spin column technology to eliminate polysaccharides phenolic compounds and enzyme inhibitors from plant tissue lysates Purified DNA is suitable for PCR restriction digestion and hybridization applications This procedure relies on the well established properties of the cationic detergent cetyltrimethyl ammonium bromide CTAB in conjun
3. E Z 96 Plant DNA DS Kit Protocol Centrifugation Protocol Materials and Equipment to be Supplied by User e Centrifuge equipped with swing bucket rotor capable of at least 3 000 x g Water baths ovens incubators capable of 65 C Vortexer 10096 ethanol Liquid nitrogen for freezing disrupting samples for fresh frozen specimens Equipment for disrupting plant tissue Before Starting Prepare VHB Buffer and DNA Wash Buffer according to Preparing Reagents section on Page 5 Set a water bath oven incubator to 65 C Heat Elution Buffer to 65 C 1 Transfer up to 10 mg dry powdered tissue or 50 mg fresh or frozen tissue to a 96 well Round well Plate provided and seal with Caps for Round well Plate Note No more than 50 mg wet weight or 10 mg dry weight starting material is recommended More or less can be used depending on results Water content and buffer absorption of samples affect optimal starting amounts 2 plant tissue following of the methods described in the Disruption of Plant Tissue section on Page 8 If homogenizing in the presence of lysis buffer with fresh tissue skip to step 4 after homogenization is complete 3 Add 700 CSPL Buffer and 20 Proteinase Solutionto each sample Seal the wells with Caps Vortex to mix thoroughly Note CSPL Buffer can be mixed with Proteinase K before use Please see the Preparing Reagents section on Page 5
4. for instructions Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields 4 Incubate at 65 C for 30 minutes Mix samples twice during incubation by briefly 10 11 12 13 14 15 16 17 10 E Z 96 Plant DNA DS Kit Protocols shaking the plate side to side Centrifuge at 3 000 6 000 x g for 10 minutes Remove and discard the caps Place the E Z 96 Homogenizer Plate on to a 96 well Square well Plate provided Transfer 550 uL cleared supernatant to the E Z 96 Homogenizer Plate Centrifuge at 3 000 6 000 x g for 5 minutes Add 5 uL Rnase A Let sit at room temperature for 5 minutes Add 525 uL RBB Buffer and 525 uL XP2 Buffer Mix thoroughly by pipetting up and down or vortexing Place the E Z 96 DNA Plate on to a new 96 well Square well Plate provided Carefully transfer 750 uL sample to the E Z 96 DNA Plate Be careful not to spill sample liquid onto the rims of the wells during the transfer Centrifuge at 3 000 5 000 x g for 5 minutes or until all the sample has passed through the HiBind membrane Discard the filtrate and reuse the 96 well Square well Plate Carefully transfer another 750 sample to the E Z 96 DNA Plate Be careful not to spill sample liquid onto the rims of the wells during the transfer Centrifuge at 3 000 5 000 x g for 5 minutes or until all the sample has passed through the HiBind memb
5. through the plate Turn off the vacuum Repeat Steps 14 16 until all the sample has been transferred to the E Z 96 DNA Plate 18 19 20 21 22 23 24 25 26 27 28 29 30 7 96 Plant DNA DS Kit Protocols Add 500 uL VHB Wash Buffer to each well Note VHB Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instructions Turn on the vacuum source to draw the VHB Wash Buffer through the plate Turn off the vacuum Add 700 uL DNA Wash Buffer to each well Note DNA Wash Buffer must be diluted with 10096 ethanol prior to use Please see Page 5 for instructions Turn on the vacuum source to draw the DNA Wash Buffer through the plate Turn off the vacuum Repeat Steps 21 23 for a second DNA Wash Buffer wash step Add 400 uL 100 ethanol to each well Turn on the vacuum source to draw the ethanol through the plate Turn off the vacuum Continue to apply the vacuum for 10 minutes after all liquid has passed through the E Z 96 DNA Plate Turn off the vacuum Discard and remove the filtrate and waste collection plate T5 31 32 33 34 35 36 37 7 96 Plant DNA DS Kit Protocols Place the E Z 96 DNA Plate upside down on a stack of paper towels and tap several times to remove any residual ethanol Note It is very important to completely dry the E Z 96 DNA Plate before elution If a swing bucket centrifuge with a 96 well plat
6. ature D1411 01 224 mL Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D1411 00 160 mL D1411 01 640 mL Guidelines for Vacuum Manifold The following is required for use with the Vacuum Protocol A Vacuum Manifold We recommend Omega Bio tek s VAC 03 Other Compatible Vacuum Manifolds Qiagen QlAvac24 Sigma AldrichVM20 Promega or manifold with standard Luer connector B Vacuum Flask C Vacuum Tubing D Vacuum Source review tables below for pressure settings Recommended Pressure mbar VAC 03 200 to 400 Conversion from millibars Multiply by Millimeters of Mercury mmHg 0 75 Kilopascals kPa 0 1 Inches of Mercury inchHg 0 0295 Tors Atmospheres atmos 0 000987 Pounds per Square Inch psi 0 0145 Illustrated Vacuum Setup ace Omega Bio tek s VAC 03 C Vacuum Tubing D Vacuum Source A Vacuum Manifold B Vacuum Flask Guidelines for Vacuum Manifold DNA Bind and Wash Setup E Z 96 DNA Plate Vacuum Manifold Top Waste Collection Vacuum Manifold Base Standard Elution Setup Optional Elution Setup E Z 96 DNA Plate E Z 96 DNA Plate Vacuum Manifold Top Vacuum Manifold Top Microplate 300 Racked Microtubes Racked Microtubes Microplate 300 Vacuum Manifold Vacuum Manifold E Eb Base Ehe Base Disruption of Plant Tissues Disrupt Samples With Commer
7. cial Homogenizers A Specimens Dried lyophilized plant tissue can be effectively disrupted and homogenized by rapid agitation in the presence of beads 1 Add one 3 4 mm stainless steel bead to each well of a 1 2 mL HTS plate 2 Close the individual tubes with Caps for Racked Microtubes 3 Place the racks or plates into the clamps of the homogenizer 4 for 60 90 seconds at 30 Hz Tissue samples are disrupted and simultaneously homogenized with the shearing and crushing action of the beads B Fresh Frozen Specimens Fresh frozen and dried plant tissue can be effectively disrupted and homogenized by rapid agitation in the presence of beads For Fresh Frozen and Lyophilized Dried Tissue 1 Add one 3 4 mm stainless steel bead to each well of a 1 2 mL HTS plate 2 Closethe individual tubes with Caps for Racked Microtubes 3 Freeze samples in liquid nitrogen Alternative to liquid nitrogen Sample can be homogenized in presence of 700 uL CSPL Buffer and 20 uL proteinase K solution for fresh samples Skip to step 4 of the E Z 96 Plant DS Protocol if homogenized in presenece of lysis buffer Complete steps 4 and 5 of disruption of plant tissue below 4 Place the racks or plates into the clamps of the homogenizer 5 Homogenize for 60 90 seconds at 30 Hz Tissue samples are disrupted and simultaneously homogenized with the shearing and crushing action of the beads 7 96 Plant DNA DS Kit Protocols
8. ction with the unique binding system to increase yields and provide high quality DNA The system eliminates the need for chloroform extractions traditionally associated with CTAB based lysis methods Samples are homogenized and lysed in a high salt buffer containing CTAB binding conditions are adjusted and DNA is purified using a E Z 96 DNA Plate Salts proteins and other contaminants are removed to yield high quality genomic DNA suitable for downstream applications such as endonuclease digestion thermal cycle amplification and hybridization applications Centrifugation Protocol Vacuum Protocol Collect Plant Tissue and Homogenize Collect Plant Tissue and Homogenize Lyse Lyse Transfer Cleared Lysate and Adjust Binding Conditions Transfer Cleared Lysate and Adjust Binding Conditions Bind and Wash 3X Bind and Wash 3X Dry Membrane Elute Kit Contents Pee ETT IET 3 e Storage and Stability All components of the E Z 96 Plant DNA DS Kit are guaranteed for at least 12 months from date of purchase when stored as follows Store RNase A at 2 8 C All other components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in VHB Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents Dilute VHB Buffer with 100 ethanol as follows and store at room temper
9. e adaptor is available centrifuge at 5 000 x g for 5 minutes to dry the plate Or if an oven incubator is available dry the plate at 65 C for 10 minutes Place the 96 well Racked Microtubes inside the base of the manifold Place the E Z 96 DNA Plate on top of the manifold Add 100 uL Elution Buffer heated to 65 C to each well Let sit at room temperature for 5 minutes Turn on the vacuum source to draw the Elution Buffer through the plate Turn off the vacuum Optional Repeat Steps 34 37 for a second elution step 38 39 16 Note 100 uL Elution Buffer is sufficient to elute up to 85 of the DNA from each well of the E Z 96 DNA Plate A second elution step with same 100 elute containing DNA reheated to 65 C will increase yield by up to 10 15 Total DNA yields vary depending on type and quantity of sample Seal the 96 well Racked Microtubes with Caps for Racked Microtubes provided Store DNA at 20 C Troubleshooting Guide Please use this guide to troubleshoot any problems that may arise For further assistance please contact the technical support staff toll free at 1 800 832 8896 Possible Problems and Suggestions Do not exceed suggested amount of starting material Problem Cause Solution Incomplete disruption of rup Completely homogenize sample starting material Decrease the amount of starting material or increase the amount of CSPL Buffer Clogged well Sample too viscous Poo
10. h tissue skip to step 4 after homogenization is complete 3 Add 700 CSPL Buffer and 20 Proteinase Solutionto each sample Seal the wells with Caps Vortex to mix thoroughly Note CSPL Buffer can be mixed with Proteinase K before use Please see the Preparing Reagents section on Page 5 for instructions Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields 13 10 11 12 13 14 15 16 17 14 7 96 Plant DNA DS Kit Protocols Incubate at 65 C for 30 minutes Mix samples twice during incubation by briefly shaking the plate side to side Centrifuge at 3 000 6 000 x g for 10 minutes Remove and discard the caps Place the E Z 96 Homogenizer Plate on to a 96 well Square well Plate provided Transfer 550 uL cleared supernatant to the E Z 96 Homogenizer Plate Centrifuge at 3 000 6 000 x g for 5 minutes Add 5 uL Rnase A Let sit at room temperature for 5 minutes Add 525 uL RBB Buffer and 525 uL XP2 Buffer Mix thoroughly by pipetting up and down or vortexing Prepare the vacuum manifold according to manufacturer s instructions Place an E Z 96 DNA Plate on the top part of the vacuum manifold Place the waste collection tray inside the base of the manifold Seal any unused wells with sealing film not provided Transfer 750 uL sample to the E Z 96 DNA Plate Turn on the vacuum source to draw the sample
11. r lysis of tissue Increase elution volume to 200 uL and incubate the plate at 65 C for 5 minutes before centrifugation DNA remains bound to column Low DNA yield Dilute SPW Wash Buffer by adding DNA washed off appropriate volume of 100 ethanol prior to use Page 5 If 550 uL lysis buffer cannot be transferred after clearing lysate by Insufficient sample centrifugation Increase volume of CSPL amount transferred af Buffer If only 350 uL could be recovered ter supernatant removal then increase amount by 200 uL 550 uL Desired amount 350 uL 200 uL additional lysis buffer amount required Repeat wash step with SPW Wash Buffer Problems in downstream Following the second wash spin ensure applications Ethanol carryover that the plate is completely dried before elution 17 18 Ordering Information The following components are available for purchase separately Call Toll free at 1 800 832 8896 Elution Buffer 100 mL Sealing Film AeraSeal Film 96 well Square well Plate 2 2 mL E Z 96 DNA Plates 10 E Z 96 Homogenizer Plates 4 x 96 E Z 96 Lysate Clearance Plates 10 x 96 Vacuum Manifold VAC 03 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen and Vacman are all trademarks of their respected companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license
12. rane 18 19 20 21 22 23 24 25 26 27 28 29 7 96 Plant DNA DS Kit Protocols Discard the filtrate and reuse the 96 well Square well Plate Add 500 uL VHB Wash Buffer to each well of the E Z 96 DNA Plate Note VHB Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instructions Centrifuge at 3 000 5 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Add 700 uL DNA Wash Buffer to each well of the E Z 96 DNA Plate Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 5 for instructions Centrifuge at 3 000 5 000 x g for 5 minutes Discard the filtrate and reuse the 96 well Square well Plate Repeat Steps 22 24 for a second DNA Wash Buffer wash step Centrifuge at 3 000 5 000 x g for 15 minutes to dry the plate Note It is important to dry the plate membrane before elution Residual ethanol may interfere with downstream applications Transfer the E Z 96 DNA Plate to new 96 well Racked Microtubes provided or a 96 well microplate not provided Add 100 uL Elution Buffer heated at 65 C to each well Incubate at 65 C for 5 minutes 11 30 31 32 33 12 7 96 Plant DNA DS Kit Protocols Centrifuge at 5 000 x g for 5 minutes Repeat Steps 28 30for a second elution step Note To maintain higher DNA concentration second elution may be performed with first eluate
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