User Manual FavorPrep Soil DNA Isolation Midi Kit
Contents
1. x g in a microcentrifuge 6 Preheat Elution Buffer or ddH2O to 60 C for elution step Brief Procedure soil sample l Lysis SDE1 70 C 10 min 1 SDE2 on ice 5 min i DNA Precipitation Isopropanol centrifuge l DNA dissolve SDE3 room Temp 2 min centrifuge SDE4 Ethanol 96 100 1 Binding centrifuge a Wash X2 centrifuge H Elution General Protocol Please Read Important Notes Before Starting Following Steps 1 Transfer up to 10 g of soil sample to a 50 ml centrifuge tube not provided 2 Weigh 2 g of Glass bead and mix with soil sample 3 Add 15 ml of SDE1 Buffer to the sample vortex at maximum speed for 5 minutes Incubate the sample at 70 C for 10 minutes and vortex the sample twice during the incubation For isolation of DNA from gram positive baceria do a further incubation at 95 C for 5 minutes 4 Cool down the sample and add 5 ml of SDE2 Buffer to the sample mix well by vortexing Incubate the sample on ice for 5 minutes 5 Cenirifuge at 2 500 x g for 5 minutes at room temperature 6 Carefully transfer the clarified lysate to a 50 ml centrifuge tube not provied And measure the volume of the clarified lysate Avoid pipetting any debris and pellet 7 Add 1 volume of isopropanol vortex to mix well centrifuge at 15 000 x g at 4 C for 30 min to precipate DNA For example If the clarified lysate volum2. FavorPrep Soil DNA Isolation Midi Kit User Manual Cat No FASOI 002 24 Preps For Research Use Only v 0905 Introduction The FavorPrep Soil DNA Isolation Midi Kit is designed for isolation of total DNA from 1 10 g of soil sample The inhibitors of the downstream PCR or enzymatic reactions will be removed with the sequent buffers system of this kit The entire procedure is not reguired the phenol chloroform extraction and can be finished within 90 min The purified DNA is ready for PCR and other downstream application Kit Contents FASOI002 24 Preps Glass Beads 55g SDE1 Buffer 200 ml X2 SDE2 Buffer 135 ml SDE3 Buffer 30 ml SDE4 Buffer 85 ml Wash Buffer concentrated 40 ml X2 Elution Buffer 120 ml SDE Midi Column 24 pcs Elution Tube 50 ml centrifuge tube 24 pcs User Manual 1 Add 160 ml ethanol 96 100 to Wash Buffer when first open Applications PCR Real Time PCR Infection disease research Sample amount Sample up to 10 g of soil sample Handing time 90 minutes Elution Volume 2 ml Important Notes 1 Buffers provided in this system contain irritants Wear gloves and lab coat when handling these buffers 2 Check SDE1 Buffer before use Warm SDE1 Buffer at 60 C for 10 minutes if any precipitate formd 3 Add 6 ml of ethanol 96 100 to Wash Buffer when first open 4 Prepare a water baths to 70 C before the operation 5 All centrifuge steps are done at full speed 14 000 rpm or 10 000
3. e is 12 ml add 12 ml of isopropanol to the clarified Itsate 8 Carefully discard the supernatant and invert the tube on the paper towel for 5 min to remove residual liquid Do not disturb the pellet 9 Add 2 ml of pre heated Elution Buffer or ddH20 vortex to dissolve the pellet completely 10 Add 1 ml of SDE3 Buffer to the sample mix well by vortexing Incubate the sample at room temperature for 3 minutes Note SDE3 Buffer must be suspended completely by vigorously vrotexing before every using Use Iml pipettor and cut off the end of 1 ml tip to make it easier for pipetting the SDE3 Buffer 11 Centrifuge at 2 500 x g at room temperature for 5 minutes 12 Carefully transfer the clarified lysate to a 15 ml or 50 ml microcentrifuge not provied And measure the volume of the clarified lysate Avoid pipetting any debris and pellet 13 Optional If RNA free DNA is required add 4 ul of 100 mg ml RNase A not provided to the sample and mix well Incubate at room temperature for 2 min 14 Add 1 volume of SDE4 Buffer and 1 volume of ethanol 96 100 to the clarified lysate mix thoroughly by pulse vortexing For example If the clarified lysate volume is 2 5 ml add 2 5 ml of SDE4 Buffer and 2 5 ml of ethanol 96 100 to the clarified Itsate 15 Place the SDE Midi Column in a 50 ml centrifuge Ex Falcon 50 ml and transfer all of the sample mixture to the SDE Midi Column 16 Centrifuge at 2 500 x g at room tem
4. perature for 2 min then discard the flow through Then place the SDE Midi Column back in 50 ml centrifuge tube 17 Add 7 ml of Wash Buffer ethanol added to SDE Midi Column Centrifuge at 2 500 x g at room temperature for 2 min then discard the flow through And repeat this step for one more time Make sure that ethanol 96 100 has been added into Wash Buffer when first open 18 19 Centrifuge at 2 500 4 000 x g at room temperature for an additional 10 min to dry the SDE Midi column It might be necessary to dry the column futher by placing the column in a vacuum oven at 70 C for 10 minutes Important step This step will avoid the residual liquid to inhibit subsequent enzymatic reactions Place SDE Midi Column into a new 50 centrifuge tube Add 1 2 ml of preheated Elution Buffer or ddH2O to the membrane center of the SDE Midi Column Stand the SDE Midi Column for 5 min at room temperature Important step For effective elution make sure that the Elution Buffer or ddH2O is dispensed onto the membrane center and is absorbed completely 20 Cenirifuge at 2 500 x g at room temperature for 2 min to elute DNA
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