E.Z.N.A.®SP Fungal DNA Mini Kit - Omega Bio-Tek
Contents
1. clumps in the solution Clumps will result in low yields Incubate at 65 C for 10 minutes Mix sample 2 to 3 times during incubation by invert ing the tube Add 140 uL SFG2 Buffer Vortex to mix thoroughly Centrifuge at gt 10 000 x g for 10 minutes Insert a Homogenizer Mini Column into a 2 mL Collection Tube Transfer supernatant to a Homogenizer Mini Column making sure not to disturb the pellet or transfer any debris Immediately centrifuge at 10 000 x g for 2 minutes Longer centrifugation does not improve yields Note The Homogenizer Mini Column will remove most remaining precipitates and cell debris but a small amount will pass through and form a pellet in the collection tube Be careful not disturb this pellet in Step 9 Carefully transfer the lysate to a new 1 5 mL microcentrifuge tube not provided making sure not to dislodge the pellet Measure the volume of the lysate for next step Note Using a set volume of lysate for each sample will eliminate the need for multiple measurements Add 1 5 volumes SFG3 Buffer For example 500 uL lysate would require 750 uL SFG3 Buffer Vortex to obtain a homogeneous mixture Note SFG3 Buffer must be diluted with 100 ethanol prior to use Please refer to Page 4 for instructions 11 E Z N A SP Fungal DNA Mini Kit Protocol Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 12 13 14 15 16 12. prior to use Please refer to Page 4 for instructions 12 E Z N A SP Fungal DNA Mini Kit Protocol Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 13 14 15 16 17 18 19 20 21 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes Centrifuge at maximum speed for 60 seconds Discard the filtrate and reuse the Collection Tube Fe a Transfer 650 uL sample from Step 11 to the HiBind DNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the collection tube Repeat Steps 13 15 until all the remaining sample has been transferred to the HiBind DNA Mini Column Transfer the column into a new 2 mL Collection Tube Add 650 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 100 ethanol prior to use Please refer to Page 4 or the bottle label for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 18 20 for a second SPW Wash Buffer wash step 22 23 24 25 26 27 28 E Z N A SP Fungal DNA Mini Kit Protocol Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is critical to remove any trace of ethanol that may otherwise interfere with downstream applications Transfer the HiBind DNA Mini Column to a nucleas
3. prolonged period of time prior to processing Samples can be dried overnight in a 45 C oven powdered and stored dry at room temperature To prepare dried samples place 25 mg of dried tissue into a microcentrifuge tube and grind using a pellet pestle Disposable Kontes pestles work well and are available from Omega Bio tek Cat SSI 1015 39 For critical work such as PCR and cloning pestles are best used a single time then soaked in a dilute bleach solution immediately after use until cleaning Disposable pestles may be autoclaved several times For standard Southern analysis the same pestle can be reused several times to grind multiple tissue samples by rinsing with ethanol and wiping the surface clean between samples A fine powder will ensure optimal DNA extraction and yield Process in sets of four to six tubes until Step 2 before starting another set Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 10 000 x g Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water bath incubator or heat block equilibrated to 65 C e Ice bucket 100 ethanol Before Starting Prepare SPW Wash Buffer and SFG3 Buffer according to the Preparing Reagents section on Page 4 Prepare an ice bucket Set an incubator heat block or water bath to 65 C Heat Elution Buffer to 65 C 1 Transfer 10 25 mg dry powdered tissue to a nuclease free 1 5 mL or 2 mL microcentrifuge tube not provided 2 Ad
4. 7 18 19 20 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes Centrifuge at maximum speed for 60 seconds Discard the filtrate and reuse the Collection Tube Fe a Transfer 650 uL sample from Step 10 to the HiBind DNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the collection tube Repeat Steps 12 14 until all the remaining sample has been transferred to the HiBind DNA Mini Column Transfer the column into a new 2 mL Collection Tube Add 650 uL SPW Wash Buffer Note SPW Wash Buffer must be diluted with 100 ethanol prior to use Please refer to Page 4 or the bottle label for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the Collection Tube Repeat Steps 17 19 for a second SPW Wash Buffer wash step 11 21 22 23 24 25 26 27 12 E Z N A SP Fungal DNA Mini Kit Protocol Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 minutes to dry the membrane Note It is critical to remove any trace of ethanol that may otherwise interfere with downstream applications Transfer the HiBind DNA Mini Column to a nuclease free 1 5 or 2 mL microcentrifuge tube Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recom
5. A Elution Buffer User Manual Storage and Stability All components of the E Z N A SP Fungal Mini Kit are stable for at least 12 months from date of purchase when stored as follows RNase A should be stored at 2 8 C All remaining components should be stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in SFG3 Buffer It is possible to dissolve such deposits by warming the solution at 37 C with gentle shaking Before Beginning Preparing Reagents 1 Dilute SFG3 Buffer with 100 ethanol as follows and store at room temperature D5542 02 160 mL 2 Dilute SPW Wash Buffer with 100 ethanol as follows and store at room temperature SS SSS 100 Ethanol to be Added Protocol Selection Guide For processing lt 25 mg powdered tissue DNA Dried Specimens yields vary depending on genome size ploidy and sample age Yields typically range from 5 50 ug per 30 mg dried tissue For processing lt 100 mg fresh or frozen tissue Fresh or Frozen Specimens j Yields typically ranges from 3 30 ug E Z N A SP Fungal DNA Mini Kit Protocol E Z N A SP Fungal DNA Mini Kit Dried Specimen Protocol This is the most robust method for isolation of total cellular mitochondrial chloroplast and genomic DNA from dried fungal samples Yields are usually sufficient for several tracks on a Southern blot for RFLP mapping Drying allows storage of field specimens for
6. Ca OMEGA Innovations in nucleic acid isolation Product Manual E Z N A SP Fungal DNA Mini Kit D5542 00 5 preps D5542 01 50 preps D5542 02 200 preps June 2013 For research use only Not intended for diagnostic testing E Z N A SP Fungal DNA Mini Kit Table of Contents Introduction and OvervieW sesssssssseeesersssssssseeeeressrssssssesse 2 Kit Contents Storage and Stability ssssecsseeceecseeeneers 3 Preparing Reagents Protocol Selection Guide s08 4 Protocol for Dried SPeCIMENS cssssecssecseessseceesecsneeseecseeeseess 5 Protocol for Fresh Frozen Spe ciMens ccsssssssssessssceesessseeees 9 Troubleshooting Guide ssssssssseeesssessssssteeseesssssssseeeserssesssss 13 Ordering atanena anne ce eee ote 14 Manual Revision June 2013 024 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview E Z N A SP Fungal Mini Kits are specially designed for rapid and reliable isolation of high quality total cellular DNA from fungal species that contain high levels of phenolic compounds and polysaccharides Up to 100 mg wet tissue or 25 mg dry tissue can be processed in less than 1 hour The system combines the reversible nucleic acid binding properties of Omega Bio tek s HiBind matrix with the speed and versatility of spin column technology to eliminate polysaccharides phenolic compounds and enzyme inhibitors from fungal tissue lysates The newly introduced ho
7. air of tweezers to fill the tube Grind the tissue using disposable Kontes pellet pestles which are available from OBI Cat SSI 1015 39 Alternatively one can allow liquid nitrogen to evaporate and then store samples at 70 C for later use For critical work such as PCR and cloning pestles are best used a single time then soaked in a dilute bleach solution immediately after use until clean Disposable pestles may be autoclaved several times For standard Southern analysis the same pestle can be reused several times to grind multiple tissue samples by rinsing with ethanol and carefully wiping the surfaces clean between samples Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 10 000 x g Nuclease free 1 5 mL or 2 mL microcentrifuge tubes Water bath incubator or heat block equilibrated to 65 C 100 ethanol Liquid nitrogen for freezing disrupting samples Before Starting Prepare SPW Wash Buffer and SFG3 Buffer according to the Preparing Reagents section on Page 4 Set an incubator heat block or water bath to 65 C e Heat Elution Buffer to 65 C 1 Transfer lt 100 mg ground fungal tissue to a nuclease free 1 5 mL or 2 mL microcentrifuge tube not provided 10 10 E Z N A SP Fungal DNA Mini Kit Protocol Add 400 uL SFG1 Buffer and 4 uL RNase A Vortex at maximum speed to mix thoroughly Note Ensure that all the samples are completely suspended and that there are no
8. d 600 uL SFG1 Buffer and 4 uL RNase A Vortex at maximum speed to mix thoroughly Note Ensure that all the samples are completely suspended and that there are no clumps in the solution Clumps will result in low yields 10 11 E Z N A SP Fungal DNA Mini Kit Protocol Incubate at 65 C for 10 20 minutes Mix sample 2 to 3 times during incubation by inverting the tube Add 210 uL SFG2 Buffer Vortex to mix thoroughly Let sit on ice for 5 minutes Centrifuge at gt 10 000 x g for 10 minutes Insert a Homogenizer Mini Column into a 2 mL Collection Tube Transfer supernatant to a Homogenizer Mini Column making sure not to disturb the pellet or transfer any debris Immediately centrifuge at 10 000 x g for 2 minutes Longer centrifugation does not improve yields Note The Homogenizer Mini Column will remove most remaining precipitates and cell debris but a small amount will pass through and form a pellet in the collection tube Be careful not disturb this pellet in Step 10 Carefully transfer the lysate to a new 1 5 mL microcentrifuge tube not provided making sure not to dislodge the pellet Measure the volume of the lysate for next step Note Using a set volume of lysate for each sample will eliminate the need for multiple measurements Add 1 5 volumes SFG3 Buffer For example 500 uL lysate would require 750 uL SFG3 Buffer Vortex to obtain a homogeneous mixture Note SFG3 Buffer must be diluted with 100 ethanol
9. e free 1 5 or 2 mL microcentrifuge tube Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller elution volumes will increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit for 3 to 5 minutes at room temperature Centrifuge at 10 000 x g for 1 minute Repeat Steps 24 26 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume Store DNA at 20 C E Z N A SP Fungal DNA Mini Kit Protocol E Z N A SP Fungal DNA Mini Kit Fresh Frozen Specimen Protocol This protocol is suitable for most fresh or frozen tissue samples that allows for more efficient recovery of DNA However due to the tremendous variation in water and polysaccharide content of fungal species sample size should be limited to lt 100 mg Best results are obtained with young tissue The method isolates sufficient DNA for several tracks on a standard Southern assay To prepare samples collect tissue in a 1 5 mL or 2 mL microcentrifuge tube and freeze by dipping in liquid nitrogen with a p
10. lution Following the second wash spin ensure that the column is dried by centrifuging 2 minutes at maximum speed 13 14 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 Homogenizer Mini Column 50 200 HCRO01 HCROO3 Homogenization Pestles 1 5 mL 10 bag 20 bags cs SSI 1015 39 SPW Wash Buffer 25 mL PDR045 RNase A 5 mL AC118 Elution Buffer 100 mL PDR048 HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license
11. mended Let sit for 3 to 5 minutes at room temperature Centrifuge at 10 000 x g for 1 minute Repeat Steps 23 25 for a second elution step Note Any combination of the following steps can be used to help increase DNA yield After adding the Elution Buffer incubate the column for 5 minutes Increase the elution volume Repeat the elution step with fresh Elution Buffer this may increase the yield but decrease the concentration e Repeat the elution step using the eluate from the first elution this may increase yield while maintaining elution volume 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 Incomplete disruption of starting material Poor lysis of tissue Low DNA yield DNA remains bound to column DNA washed off SPW Wash Buffer must be at room Salt carryover temperature Problems in downstream applications Ethanol carryover For both dry and fresh samples obtain a fine homogeneous powder before adding SFG1 Buffer Decrease amount of starting material or increase the amount of SFG1 Buffer and SFG2 Buffer Increase elution volume to 200 uL and incubate the column at 65 C for 5 minutes before centrifugation Dilute SPW Wash Buffer by adding the appropriate volume of 100 ethanol prior to use see Page 4 for instructions So
12. mogenization columns provide a fast and easy tool for sample homogenization Purified DNA is suitable for PCR restriction digestion and hybridization applications There are no organic extractions thus reducing plastic waste and hands on time to allow multiple samples to be processed in parallel If using the E Z N A SP Fungal Mini Kit for the first time please read this booklet to become familiar with the procedures Dry or fresh fungal tissue is disrupted and lysed in a specially formulated buffer containing detergent Proteins polysaccharides and cellular debris are subsequently precipitated Binding conditions are adjusted and the sample is transferred to a HiBind DNA Mini Column Two rapid wash steps remove trace contaminants such as residual polysaccharides and pure DNA is eluted in Elution Buffer Purified DNA can be directly used in downstream applications without the need for further purification New in this Edition e This manual has been edited for content and redesigned to enhance user readability e Equilibration Buffer is no longer included with this kit An optional Column Equilibration Protocol has been added to the protocol for your convenience Equilibration Buffer is replaced with 3M NaOH provided by the user Kit Contents D5542 00 D5542 01 D5542 02 Purifications HiBind DNA Mini Columns 2 mL Collection Tubes Homogenizer Mini Column SFG1 Buffer SFG2 Buffer SFG3 Buffer SPW Wash Buffer RNase
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