E.Z.N.A.®Fungal DNA Mini Kit - Omega Bio-Tek
Contents
1. 11 12 13 E Z N A Fungal DNA Mini Kit Protocols Incubate at 65 C for 10 min Mix sample twice during incubation by inverting tube Add 180 uL FG2 Buffer Vortex to mix thoroughly Let sit on ice for 5 minutes Centrifuge at 10 000 x g for 10 minutes Transfer supernatant to a new microcentrifuge tube making sure not to disturb the pellet or transfer any debris Add 0 7 volumes isopropanol Vortex to precipitate DNA Note In most cases 700 uL supernatant can easily be removed This will require 490 uL isopropanol Note that depending on the sample the volume of supernatant may vary After transferring to a fresh tube measure the volume and add the correct amount of isopropanol This step will remove much of the polysaccharide content and improves spin column performance by increasing DNA binding capacity and hence yield in the steps that follow No incubation is required after addition of isopropanol Immediately centrifuge at 10 000 x g for 2 minutes Longer centrifugation does not improve yields Aspirate and discard the supernatant making sure not to dislodge the DNA pellet Invert the microcentrifuge tube on a paper towel for 1 minute to allow residual liquid to drain It is not necessary to dry the DNA pellet Add 300 uL sterile deionized water heated to 65 C Vortex to resuspend the pellet Note A brief incubation at 65 C may be necessary to effectively dissolve the DNA Add 4 uL RNase A Vortex t2. 10 E Z N A Fungal DNA Mini Kit Protocols Incubate at 65 C for 10 minutes Mix sample twice during incubation by inverting tube Add 140 uL FG2 Buffer Vortex to mix thoroughly Let sit on ice for 5 minutes Centrifuge at 10 000 x g for 10 minutes Transfer supernatant to a new microcentrifuge tube making sure not to disturb the pellet or transfer any debris Add 0 7 volumes isopropanol Vortex to precipitate DNA Note In most cases 600 uL supernatant can easily be removed This will require 420 uL isopropanol Note that depending on the sample the volume of supernatant may vary After transferring to a fresh tube measure the volume and add the correct amount of isopropanol This step will remove much of the polysaccharide content and improves spin column performance by increasing DNA binding capacity and hence yield in the steps that follow No incubation is required after addition of isopropanol Immediately centrifuge at 10 000 x g for 2 minutes Longer centrifugation does not improve yields Aspirate and discard the supernatant making sure not to dislodge the DNA pellet Invert the microcentrifuge tube on a paper towel for 1 minute to allow residual liquid to drain It is not necessary to dry the DNA pellet Add 300 uL sterile deionized water heated to 65 C Vortex to resuspend the pellet Note A brief incubation at 65 C may be necessary to effectively dissolve the DNA Add 4 uL RNase A Vortex to mix thoro
3. 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 significantly increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit for 3 to 5 minutes Centrifuge at 10 000 x g for 1 minute Repeat Steps 26 28 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 Fungal DNA Mini Kit Protocols E Z N A Fungal DNA Mini Kit Protocol Short Protocol This simplified method allows rapid isolation of DNA from fresh frozen or dried specimens for use in PCR reactions The procedure limits the amount of starting material so that DNA yields will generally be lower than those obtained with the previous protocols The short protocol is not recommended for Southern analysis or cloning work as in most cases there will be insufficient material Materials and Equipment to be Supplied by User M
4. 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 recommended Let sit for 3 to 5 minutes Centrifuge at 10 000 x g for 1 minute Repeat Steps 26 28 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 Fungal DNA Mini Kit Protocols E Z N A Fungal DNA Mini Kit Protocol Fresh or Frozen Specimens This protocol is suitable for most fresh or frozen tissue samples However due to the tremendous variation in water and polysaccharide content of various fungi sample size should be limited to lt 200 mg The method isolates sufficient DNA for several tracks on a standard Southern assay To prepare samples collect tissue in a 1 5 or 2 mL microcentrifuge tube and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube Grind
5. interfere with downstream applications Turn off the vacuum source 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 significantly increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit for 3 to 5 minutes Centrifuge at 10 000 x g for 1 minute 15 16 E Z N A Fungal DNA Mini Kit Protocols Repeat Steps 12 14 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 e Increase the elution volume e 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 19 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 Following precipitation with FG2 Buffer Debris carryover make sure no particulate material is transferred DNA pellet not In protocols A and B ensure that DNA is completely dissolved dissolved in water before adding FG3 Buffer before applying samp
6. the tissue using disposable Kontes pellet pestles which are available from Omega Bio tek 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 Cat SSI 1210 00 or SSI 1310 00 Water bath capable of 65 C Pestles for grinding tissue Cat SSI 1015 39 Sterile deionized water e lsopropanol 100 ethanol e Optional 3M NaOH Before Starting e Prepare the DNA Wash Buffer according to the instructions on Page 4 Heat the sterile deionized water and Elution Buffer to 65 C Prepare an ice bucket 1 Prepare 100 mg tissue in a 1 5 or 2 mL microcentrifuge tube 2 Add 600 uL FG1 Buffer Vortex vigorously to mix Make sure to disperse all clumps Note Process in sets of four to six tubes grind add FG1 Buffer then proceed to Step 3 before starting another set Do not exceed 200 mg tissue 9 10 11 12 13
7. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A Fungal DNA Mini Kit D3390 00 5 preps D3390 01 50 preps D3390 02 200 preps May 2013 For research use only Not intended for diagnostic testing E Z N A Fungal DNA Mini Kit Table of Contents Introduction and OVEFVIEW ssccsscescssecsseccsecssecnsecseecneceneessees 2 Kit Contents Storage and Stability secsecsseessecseeeseers 3 Before BeginniNg sssssesesesssssessssssssssssssssessseneesesssessensseeneeeereeees 4 Dried Specimen ProtoCoOl e ssssseeesesssssssssteesersssssssssssseessessnsssesse 5 Fresh or Frozen Specimen Protocol ccsssecssscscsseeseesesseeees 9 Short Protocole aE 13 Vacuum Spin ProtoCol sssssesssssseerssssssssesseeesesssssssssereseessnsssss 17 Troubleshooting Guide sessssssseresssesssssseeesersssssssseeeserssesssss 20 Manual Revision May 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview Introduction E Z N A Fungal DNA Mini Kit allows for the rapid and reliable isolation of high quality total cellular DNA from a wide variety of fungal species and tissues Up to 200 mg of wet tissue or up to 50 mg dry tissue can be processed in less than 1 hour The system combines the reversible nucleic acid binding properties of Omega s HiBind matrix with the speed and versatility of spin columns to eliminate polysaccharides phenolic compounds and enzym
8. e inhibitors from fungal tissue lysates 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 Overview If using the E Z N A Fungal DNA 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 Contaminants are further removed by isopropanol precipitation of DNA Binding conditions are then adjusted and the sample is applied to an HiBind DNA Mini Column Two rapid wash steps remove trace contaminants such as residual polysaccharides and pure DNA is eluted in water or low ionic strength buffer Purified DNA can be directly used in downstream applications without the need for further purification New in this Edition 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 Purifications HiBind DNA Mini Columns 2 mL Collection Tubes FG1 Buffer FG2 Buffer FG3 Buffer RNase A DNA Wash Buffer Kit Contents D3390 00 D3390 01 D3390 02 50 200 00 00 v Paes Rees rea Elutio
9. icrocentrifuge capable of at least 10 000 x g Nuclease free 2 mL microcentrifuge tubes Cat SSI 1310 00 Water bath capable of 65 C e lsopropanol 100 ethanol Liquid nitrogen B mercaptoethanol e Optional 3M NaOH e Optional Sterile deionized water Before Starting Prepare the DNA Wash Buffer according to the instructions on Page 4 Heat the sterile deionized water and Elution Buffer to 65 C For dried specimens use a maximum of 10 mg ground tissue For fresh frozen specimens use a maximum of 40 mg ground tissue Prepare an ice bucket 1 Prepare tissue in a 2 mL microcentrifuge tube 2 Add 600 uL FG1 Buffer and 5 uL RNase A Vortex vigorously to mix Make sure to disperse all clumps 3 Let sit for 1 minute 4 Add 10 uL B mercaptoethanol Vortex to mix thoroughly 5 Incubate at 65 C for at least 5 minutes Mix sample once during incubation by inverting tube 13 10 11 E Z N A Fungal DNA Mini Kit Protocols Add 140 uL FG2 Buffer Vortex to mix thoroughly Let sit on ice for 5 minutes Centrifuge at 10 000 x g for 10 minutes Transfer supernatant to a new microcentrifuge tube making sure not to disturb the pellet or transfer any debris Note In most cases 600 uL supernatant can easily be removed The volume of supernatant will vary and is usually lower with dried samples Add 0 5 volumes FG3 Buffer and 1 volume 100 ethanol Vortex to mix thoroughly Note A precipitate may for
10. le and ethanol This may need repeated Clogged to column incubation at 65 C with vortexing column In protocol C do not exceed suggested amount of starting material Alternatively increase the amounts of FG1 and FG2 Buffers and use two or more columns per sample Sample too viscous Incomplete precipitation following addition of FG2 Buffer Increase speed or time of centrifugation after addition of FG2 Buffer For both dry and fresh samples obtain a fine homogeneous powder before adding FG1 Buffer eee ee Decrease amount of starting material or Y increase amount of FG1 and FG2 Buffers Increase elution volume to 200 uL and incubate column at 65 C for 5 minutes before centrifugation Dilute DNA Wash Buffer by adding the DNA washed off appropriate volume of ethanol prior to use Page 4 Incomplete disruption of starting material Low DNA yield DNA remains bound to column DNA Wash Buffer must be at room Salt carryover Problems in temperature downstream A Following the second wash step ensure applications Ethanol carryover that the column is dried by centrifuging 2 minutes at maximum speed HiBind E Z N A and MicroElute are registered trademarks of Omega Bio tek Inc Qiagen QlAvac and Vacman are all trademarks of their respective companies PCR is a patented process of Hoffman La Roche Use of the PCR process requires a license 20
11. m upon addition of ethanol it will not interfere with DNA isolation Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 12 13 14 15 14 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 PWN gt Transfer 800 uL sample from Step 10 including any precipitate that may have formed 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 of the sample has been transferred to the HiBind DNA Mini Column 16 17 18 19 20 21 22 23 24 25 E Z N A Fungal DNA Mini Kit Protocols Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube Add 750 uL DNA Wash Buffer Note DNA 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 DNA Wash Buffer wash step 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
12. mmended for processing of gt 50 mg tissue 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 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 Cat SSI 1210 00 or SSI 1310 00 Water bath capable of 65 C Pestles for grinding tissue Cat SSI 1015 39 Sterile deionized water e lsopropanol 100 ethanol e Optional 3M NaOH Before Starting e Prepare the DNA Wash Buffer according to the instructions on Page 4 Heat the sterile deionized water and Elution Buffer to 65 C Prepare an ice bucket 1 Prepare 10 50 mg powdered dry tissue in a 1 5 or 2 mL microcentrifuge tube 2 Add 800 uL FG1 Buffer Vortex vigorously to mix Make sure to disperse all clumps Note Process in sets of four to six tubes grind add FG1 Buffer then proceed to Step 3 before starting another set Do not exceed 50 mg dried tissue 10
13. n Buffer User Manual Storage and Stability All components of the E Z N A Fungal Mini Kit are stable for at least 24 months from date of purchase when stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in FG3 Buffer It is possible to dissolve such deposits by warming the solution at 37 C Before Beginning Preparing Reagents Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature D3390 02 100 mL per bottle Protocol Selection Guide For processing lt 50 mg powdered tissue Yield is Dried Specimens sufficient for several tracks on Southern assay For processing lt 200 mg fresh or frozen tissue Fresh or Frozen Specimens Yield is similar to that for dried specimens ShortProtocol 13 Rapid protocol for dried or fresh samples Yield is sufficient for PCR E Z N A Fungal DNA Mini Kit Protocols E Z N A Fungal DNA Mini Kit Protocol Dried Specimens This is the most robust method for isolation of total cellular mitochondrial chloroplast and genomic DNA Yields are usually sufficient for several tracks on a Southern blot for RFLP mapping Drying allows storage of field specimens for 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 50 mg of dried tissue into a microcentrifuge tube 2 mL tubes are reco
14. 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 significantly increase DNA concentration but decrease yield Elution volumes greater than 200 uL are not recommended Let sit for 3 to 5 minutes Centrifuge at 10 000 x g for 1 minute 15 26 27 16 E Z N A Fungal DNA Mini Kit Protocols 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 e Increase the elution volume e 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 Fungal DNA Mini Kit Protocols E Z N A Fungal DNA Mini Kit Protocol Vacuum Spin Protocol Note Please read through previous sections of this manual before using this protocol Materials and Equipment to be Supplied by User Vacuum Manifold Cat VAC 08 e Microcentrifuge capable of at least 10 000 x g Nuclease free 2 mL microcentrifuge tubes Cat SSI 1310 00 e Water bath capable of 65 C Sterile deionized water e lsopropanol 100 ethanol e Optional 3M NaOH Before Starting Prepare the DNA Wash Buffe
15. o mix thoroughly 14 15 E Z N A Fungal DNA Mini Kit Protocols Add 150 uL FG3 Buffer and 300 uL 100 ethanol Vortex to mix thoroughly Note A precipitate may form upon addition of ethanol it will not interfere with DNA isolation Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 16 17 18 19 20 21 22 23 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 RWN gt Transfer the entire sample including any precipitate that may have formed to the HiBind DNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and the Collection Tube Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube Add 750 uL DNA Wash Buffer Note DNA 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 20 22 for a second DNA Wash Buffer wash step 24 25 26 27 28 29 30 E Z N A Fungal DNA Mini Kit Protocols 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
16. r according to the instructions on Page 4 Heat the sterile deionized water and Elution Buffer to 65 C For dried specimens use a maximum of 10 mg ground tissue For fresh frozen specimens use a maximum of 40 mg ground tissue Prepare an ice bucket 1 Complete Steps 1 14 of the Dried Specimens protocol on Page 5 Steps 1 14 of the Fresh or Frozen Specimens protocol on Page 9 or Steps 1 10 of the Short Protocol on Page 13 2 Prepare the vacuum manifold according to manufacturer s instructions and connect the HiBind DNA Mini Column to the manifold Optional Protocol for Column Equilibration Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes Apply the vacuum until all the buffer has passed through the membrane Turn off the vacuum source fe Wo NS 3 Transfer the sample to the HiBind DNA Mini Column 17 10 11 12 13 14 18 E Z N A Fungal DNA Mini Kit Protocols Apply the vacuum until all the sample has passed through the membrane Turn off the vacuum source Add 750 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol Please see the instructions on Page 4 Apply the vacuum until all the buffer has passed through the membrane Repeat Steps 6 7 for a second DNA Wash Buffer wash step Continue to apply maximum vacuum for an additional 10 minutes to dry the membrane Note It is critical to dry the membrane Residual ethanol may
17. ughly 14 15 E Z N A Fungal DNA Mini Kit Protocols Add 150 uL FG3 Buffer and 300 uL 100 ethanol Vortex to mix thoroughly Note A precipitate may form upon addition of ethanol it will not interfere with DNA isolation Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 16 17 18 19 20 21 22 23 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 RWN gt Transfer the entire sample including any precipitate that may have formed to the HiBind DNA Mini Column Centrifuge at 10 000 x g for 1 minute Discard the filtrate and the Collection Tube Transfer the HiBind DNA Mini Column to a new 2 mL Collection Tube Add 750 uL DNA Wash Buffer Note DNA 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 20 22 for a second DNA Wash Buffer wash step 11 24 25 26 27 28 29 30 12 E Z N A Fungal DNA Mini Kit Protocols 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
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