E.Z.N.A.®HP Fungal DNA Kit
Contents
1. Repeat Steps 26 27 for a second elution step Note This step may be performed using a new 1 5 mL microcentrifuge tube to maintain a higher DNA concentration in the first eluate Store DNA at 20 C 15 E Z N A HP Fungal DNA Kit Protocols E Z N A HP Fungal DNA Kit Vacuum Protocol Note Please read through previous section of this book before using this protocol Materials and Equipment to be Supplied by User Vacuum Manifold Centrifuge with a swing bucket rotor capable of 3 000 x g Waterbath capable of 65 C 100 ethanol Optional sterile deionized water Before Starting Prepare DNA Wash Buffer according to the Preparing Reagents section on Page 4 Prepare a mixture of chloroform isoamyl alcohol 24 1 Heat Elution Buffer to 65 C Complete Steps 1 7 of either the Dried or Fresh Frozen Tissue Protocols Pages 6 and 9 respectively Prepare the vacuum manifold according to manufacturer s instructions Connect the HiBind DNA Mini Column to the vacuum manifold Optional Protocol for Column Equilibration 16 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes at room temperature Turn on the vacuum to draw the NaOH through the column Turn off the vacuum BW NCS Transfer the cleared supernatant from Step 7 of the Dried or Fresh Frozen Protocols Pages 7 and 10 respectively by CAREFULLY aspirating it into the HiBind DNA Mini Column Be careful not to disturb the pellet and t2. Vortex to obtain a homogeneous mixture Note A precipitate may form upon addition of ethanol it will not interfere with DNA isolation Optional This is the point to start the optional vacuum protocol See Page 16 for details 8 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 10 11 12 10 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes at room temperature Centrifuge at maximum speed for 20 seconds Discard the filtrate and reuse the Collection Tube AWNa 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 into a new 2 mL Collection Tube 13 14 15 16 17 18 19 E Z N A HP Fungal DNA Kit Protocols Add 650 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute Discard the filtrate and reuse the collection tube Repeat Steps 13 15 for a second DNA Wash Buffer wash step Centrifuge the empty column at maximum speed for 2 minutes Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA M
3. 10 minutes Carefully aspirate the top aqueous phase to a new 15 mL microcentrifuge tube making sure not to disturb the organic phase or transfer any debris Add 0 7 volumes isopropanol Vortex to mix thoroughly Immediately centrifuge at 3 000 x g for 20 minutes Longer centrifugation does not improve yield Carefully aspirate and discard the supernatant making sure not to dislodge the DNA pellet Place inverted centrifuge tube on a paper towel for 1 minute to allow residual liquid to drain It is not necessary to dry the DNA pellet Add 400 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 20 uL RNase A 20 mg mL Vortex to mix thoroughly 13 13 E Z N A HP Fungal DNA Kit Protocols Add 200 uL CXD Buffer and 400 uL 100 ethanol Vortex to obtain a homogeneous mixture Note A precipitate may form upon addition of ethanol it will not interfere with DNA isolation Optional This is the point to start the optional vacuum protocol See Page 16 for details 14 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration T5 16 17 18 19 20 21 14 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes at room temperature Centrifuge at maximum speed for 20 seconds Discard the filtrate and reuse the Collecti
4. Bio tek or its distributors for ordering information Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 20 000 x g e Centrifuge with a swing bucket rotor capable of 3 000 x g Vortexer Nuclease free 1 5 mL and 2 mL microcentrifuge tubes e Nuclease free 15 mL and 50 mL centrifuge tubes e Waterbath capable of 65 C Chloroform e Isoamyl alcohol e lsopropanol 100 ethanol e RNase A stock solution at 20 mg mL e Optional 2 mercaptoethanol e Optional sterile deionized water Before Starting Prepare DNA Wash Buffer according to the Preparing Reagents section on Page 4 Prepare a mixture of chloroform isoamyl alcohol 24 1 Heat Elution Buffer to 65 C Note Follow suggestions for preparation of dried or fresh samples as outlined in Sections A and B Pages 4 and 6 respectively Note the following limitations on sample size Dry Samples use a maximum of 200 mg ground tissue Fresh Samples use a maximum of 400 mg fresh frozen ground tissue 12 10 11 12 E Z N A HP Fungal DNA Kit Protocols Transfer ground sample to a 15 mL centrifuge tube Add 9 mL CFL Buffer Vortex to mix thoroughly Optional Add 10 uL 2 mercaptoethanol per 1 mL CFL Buffer Vortex to mix thoroughly Make sure to disperse all clumps Let sit at room temperature for 60 minutes Add 4 5 mL chloroform isoamyl alcohol 24 1 Vortex to mix thoroughly Centrifuge at 3 000 x g for
5. Ca OMEGA Innovations in nucleic acid isolation bio tek Product Manual E Z N A HP Fungal DNA Kit D3195 00 5 preps D3195 01 50 preps May 2013 For research use only Not intended for diagnostic testing E Z N A HP Fungal DNA Kit Table of Contents Introduction and OVEFVIEW cscssccsscsscsseccecssecnseessecneceneerses 2 Kit Contents Storage and Stability secsecsseeceecseeeneees 3 Preparing Reagents essseesssseeseseerssseeesseeessscesseseossssossesossesesssss 4 Processing Fungal SamMpleS ssesssssssseeesersssssssssssstessessnsssesse 5 Protocol for Dried SamMpleS ssssssssssssseeserssssssssssssseesnssssssesss 6 Protocol for Fresh Frozen SaMples scsscsesssessesscsnssseeseesses 9 Protocol for Samples with Lower DNA Content 12 VACUUM PYOLOCO savccscriersscstxtermeanrincuntentmannenmanans 16 Troubleshooting Guide ssesssssssersessssssssseeeserssssssssreeseesensssss 19 Order Gennin a E RERA 20 Manual Revision May 2013 02 OMEGA bio tek Innovations in nucleic acid isolation Introduction and Overview The E Z N A High Performance HP Fungal DNA Kit is designed for efficient recovery of genomic DNA up to 60 kb in size from fresh and dried fungal tissue samples rich in polysaccharides or having lower DNA content Up to 100 mg wet tissue or 30 mg dry tissue can be processed in less than 1 hour The system combines the reversible nucleic acid binding proper
6. ations DNA washed off Following the second wash step centrifuge Ethanol carryover the column for 2 minutes at maximum speed to completely dry the matrix 19 20 Ordering Information The following components are available for purchase separately Call Toll Free at 1 800 832 8896 DNA Wash Buffer 100 mL PSO10 Elution Buffer 100 mL PDRO48 CXD Buffer 100 mL PDO79 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
7. ecimens Drying allows storage of field specimens for prolonged periods 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 dried tissue into a 2 mL microcentrifuge tube and grind using a pellet pestle Disposable Kontes pestles work well and are available from Omega Bio tek Cat SSI 1014 39 amp 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 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 wiping the surface clean between samples A fine powder will ensure optimal DNA extraction and yield Processing Fresh Frozen Specimens To prepare fresh or frozen samples collect tissue in a 1 5 mL or 2 mL microcentrifuge tube and freeze by dipping in liquid nitrogen with a pair of tweezers to fill the tube Grind the tissue using disposable Kontes pellet pestles 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 analys
8. gal DNA Kit Protocols Add 600 uL chloroform isoamyl alcohol 24 1 Vortex to mix thoroughly Centrifuge at gt 10 000 x g for 10 minutes Carefully aspirate 300 uL aqueous phase top to a new 1 5 mL microcentrifuge tube making sure not to disturb the organic phase or transfer any debris Add 150 uL CXD Buffer and 300 uL 100 ethanol Vortex to obtain a homogeneous mixture Note A precipitate may form upon addition of ethanol it will not interfere with DNA isolation Optional This is the point to start the optional vacuum protocol See Page 16 for details 8 Insert a HiBind DNA Mini Column into a 2 mL Collection Tube Optional Protocol for Column Equilibration 10 11 12 Add 100 uL 3M NaOH to the HiBind DNA Mini Column Let sit for 4 minutes at room temperature Centrifuge at maximum speed for 20 seconds Discard the filtrate and reuse the Collection Tube FWN 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 Insert the HiBind DNA Mini Column into a new 2 mL Collection Tube 13 14 15 16 17 18 19 E Z N A HP Fungal DNA Kit Protocols Add 650 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute Discard the fil
9. hat no cellular debris is transferred to the HiBind DNA Mini Column 10 11 12 13 14 E Z N A HP Fungal DNA Kit Protocols Turn on the vacuum source to draw the sample through the column Turn off the vacuum Add 750 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Turn on the vacuum source to draw the buffer through the column Turn off the vacuum Repeat Steps 7 9 for a second DNA Wash Buffer wash step Transfer the HiBind DNA Mini Column into a new 2 mL Collection Tube Centrifuge the empty column at maximum speed for 2 minutes Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller volumes will significantly increase DNA concentration but result in lower yields Using more than 200 uL for elution is not recommended 17 E Z N A HP Fungal DNA Kit Protocols Optional Incubate the HiBind DNA Mini Column at 60 70 C for 5 minutes This step may increase yield 15 Centrifuge at maximum speed for 1 minute 16 Repeat Steps 26 27 for a second elution step Note This step may be performed using a new 1 5 mL microcentrifuge tube to maintain a higher DNA concent
10. ini Column to a clean 1 5 mL microcentrifuge tube not provided Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller volumes will significantly increase DNA concentration but result in lower yields Using more than 200 uL for elution is not recommended Optional Incubate the HiBind DNA Mini Column at 60 70 C for 5 minutes This step may increase yield 20 21 22 Centrifuge at maximum speed for 1 minute Repeat Steps 18 19 for a second elution step Note This step may be performed using a new 1 5 mL microcentrifuge tube to maintain a higher DNA concentration in the first eluate Store DNA at 20 C 11 E Z N A HP Fungal DNA Kit Protocols E Z N A HP Fungal DNA Kit Protocol for Samples with Lower DNA Content This modified method allows rapid isolation of DNA from fresh frozen or dried specimens for sample types with lower DNA content or when larger yields are essential The procedure increases the amount of starting material so that DNA yields will generally be higher than those obtained with the previous protocols Yields vary according to sample size and whether dried or fresh Between 2 10 pg restrictable DNA can usually be obtained with this method Important The buffer supplies with this kit is designed for standard protocols on Pages 6 and 9 Additional buffer will be required for this protocol Buffers can be purchase separately from Omega Bio tek please contact Omega
11. is 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 E Z N A HP Fungal DNA Kit Protocols E Z N A HP Fungal DNA Kit Protocol for Dried Samples Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 20 000 x g Nuclease free 1 5 mL and 2 mL microcentrifuge tubes Waterbath capable of 65 C Vortexer Chloroform lsoamyl alcohol lsopropanol 100 ethanol RNase A stock solution at 20 mg mL Optional 2 mercaptoethanol Optional sterile deionized water Before Starting Prepare DNA Wash Buffer according to the Preparing Reagents section on Page 4 Prepare a mixture of chloroform isoamyl alcohol 24 1 Heat Elution Buffer to 65 C Transfer 10 50 mg powdered dried tissue to a 2 mL microcentrifuge tube Tip Process in sets of four to six tubes grind add CFL Buffer and 2 mercaptoethanol and proceed to Step 3 before starting another set Initially do not exceed 50 mg dried tissue Amount can be increased according to results Add 600 uL CFL Buffer Vortex to mix thoroughly Optional Add 10 uL 2 mercaptoethanol Vortex to mix thoroughly Make sure to disperse all clumps Optional If necessary add 2 uL RNase A to the lysate before Step 3 incubation step to remove the RNA Incubate at 65 C for 30 minutes Invert the tube twice during incubation to mix the sample E Z N A HP Fun
12. on Tube RW a Transfer 700 uL sample from Step 13 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 15 17 until all of the remaining sample including any precipitates that may have formed has been transferred to the HiBind DNA Mini Column Transfer the HiBind DNA Mini Column into a new 2 mL Collection Tube Add 650 uL DNA Wash Buffer Note DNA Wash Buffer must be diluted with 100 ethanol prior to use Please see Page 4 for instructions Centrifuge at 10 000 x g for 1 minute 22 23 24 25 26 E Z N A HP Fungal DNA Kit Protocols Discard the filtrate and reuse the collection tube Repeat Steps 20 22 for a second DNA Wash Buffer wash step Centrifuge the empty column at maximum speed for 2 minutes Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller volumes will significantly increase DNA concentration but result in lower yields Using more than 200 uL for elution is not recommended Optional Incubate the HiBind DNA Mini Column at 60 70 C for 5 minutes This step may increase yield 27 28 29 Centrifuge at maximum speed for 1 minute
13. ptional Column Equilibration Protocol has been added to the protocol for your convenience e Equilibration Buffer is replaced with 3M NaOH provided by the user Kit Contents aa o or mucan w w Storage and Stability All of the E Z N A HP Fungal DNA Kit components are guaranteed for at least 12 months from the date of purchase when stored at room temperature During shipment or storage in cool ambient conditions precipitates may form in CFL Buffer and CXD Buffer Dissolve such deposits by warming the solution at 37 C and gently shaking Preparing Reagents 1 Dilute DNA Wash Buffer with 100 ethanol as follows and store at room temperature 2 Prepare a 20 mg mL RNase A stock solution and aliquot Store each aliquot at 20 C and thaw before use Each sample will require 20 uL RNase A Processing Fungal Samples Choose the most appropriate protocol to follow Procedures are described for each of dried and fresh frozen specimens In addition a short protocol is given for the isolation of DNA for PCR reactions Dry Specimens Page 6 For processing 50 mg powdered tissue DNA yields range from 50 ug to more than 100 ug per 100 mg dry tissue Fresh Frozen Specimens Page 9 For processing lt 200 mg fresh or frozen tissue Yields are similar to dry specimens Lower DNA content samples Page 12 For processing up to 200 mg dried or 450 mg fresh or frozen tissue Yields are similar to dry specimens Processing Dry Sp
14. ration in the first eluate 17 Store DNA at 20 C 18 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 extraction with chloroform Debris carryover isoamyl alcohol make sure no particulate material is transferred In the Protocol for Samples with Lower DNA pellet not DNA Content ensure that DNA is dissolved completely dissolved in water before adding CXD Buffer before applying sample and ethanol This may need repeated to column incubations at 65 C Vortex during the incubations Clogged column Do not exceed suggested amount of starting material Alternatively increase the amounts of CFL and CXD Buffers and use two or more columns per sample For both dry and fresh samples obtain a fine homogeneous powder before adding CFL Buffer Sample too viscous Incomplete disruption of starting material Decrease the amount of starting material Poor lysis of sample or increase the amount of CFL Buffer Low DNA chloroform isoamyl alcohol and CXD Buffer yield Increase the elution volume to 200 uL and DNA remains bound to incubate the column at 65 C for 5 minutes column before centrifugation Prepare DNA Wash Buffer according to the Preparing Reagents section on Page 4 DNA Wash Buffer must be at room Salt carryover Problems in temperature downstream applic
15. rifuge tubes Waterbath capable of 65 C e Vortexer Chloroform Isoamyl alcohol e lsopropanol 100 ethanol e RNase A stock solution at 20 mg mL e Optional 2 mercaptoethanol e Optional sterile deionized water Before Starting Prepare DNA Wash Buffer according to the Preparing Reagents section on Page 4 Prepare a mixture of chloroform isoamyl alcohol 24 1 Heat Elution Buffer to 65 C 1 Transfer 100 mg ground fresh frozen tissue to a 2 mL microcentrifuge tube Tip Process in sets of four to six tubes grind add CFL Buffer and 2 mercaptoethanol and proceed to Step 3 before starting another set Initially do not exceed 100 mg tissue Amount can be increased up to 200 mg according to results 2 Immediately add 500 uL CFL Buffer Vortex to mix thoroughly Optional Add 10 uL 2 mercaptoethanol Vortex to mix thoroughly Make sure to disperse all clumps Optional If necessary add 2 uL RNase A to the lysate before Step 3 incubation step to remove the RNA E Z N A HP Fungal DNA Kit Protocols Incubate at 65 C for 15 minutes Invert the tube twice during incubation to mix the sample Add 800 uL chloroform isoamyl alcohol 24 1 Vortex to mix thoroughly Centrifuge at gt 10 000 x g for 5 minutes Carefully aspirate 300 uL aqueous phase top to a new 1 5 mL microcentrifuge tube making sure not to disturb the organic phase or transfer any debris Add 150 uL CXD Buffer and 300 uL 100 ethanol
16. ties of the HiBind matrix with the speed and versatility of spin column technology to eliminate polysaccharides phenolic compounds and enzyme inhibitors from fungal tissue lysates Purified DNA is suitable for PCR restriction digestion and hybridization techniques 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 HP Fungal DNA Kit for the first time please read this booklet to become familiar with the procedures This procedure relies on the well established properties of the cationic detergent cetyltrimethyl ammonium bromide CTAB in conjunction with the selective DNA binding of Omega Bio tek s HiBind matrix to purify high quality DNA Samples are homogenized and lysed in a high salt buffer containing CTAB and extracted with chloroform to remove polysaccharides and other components that interfere with many routine DNA isolations and downstream applications Binding conditions are adjusted and DNA is further purified using HiBind DNA Mini Columns In this way 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 New in this Edition This manual has been edited for content and redesigned to enhance user readability e Equilibration Buffer is no longer included with this kit An o
17. trate and reuse the collection tube Repeat Steps 13 15 for a second DNA Wash Buffer wash step Centrifuge the empty column at maximum speed for 2 minutes Note It is important to dry the HiBind DNA Mini Column matrix before elution Residual ethanol may interfere with downstream applications Transfer the HiBind DNA Mini Column to a clean 1 5 mL microcentrifuge tube not provided Add 100 uL Elution Buffer or sterile deionized water heated to 65 C Note Smaller volumes will significantly increase DNA concentration but result in lower yields Using more than 200 uL for elution is not recommended Optional Incubate the HiBind DNA Mini Column at 60 70 C for 5 minutes This step may increase yield 20 21 22 Centrifuge at maximum speed for 1 minute Repeat Steps 18 19 for a second elution step Note This step may be performed using a new 1 5 mL microcentrifuge tube to maintain a higher DNA concentration in the first eluate Store DNA at 20 C E Z N A HP Fungal DNA Kit Protocols E Z N A HP Fungal DNA Kit Protocol for Fresh Frozen Samples This protocol is suitable for most fresh frozen tissue samples allowing more efficient recovery of DNA Due to the tremendous variation in water and polysaccharide content of fungi sample size should be limited to lt 200 mg Materials and Equipment to be Supplied by User Microcentrifuge capable of at least 20 000 x g Nuclease free 1 5 mL and 2 mL microcent
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