Invisorb Genomic DNA Kit II User manual
Contents
1. Ordering information S oowoo Nonon ar w 11 12 13 14 2 Invisorb Genomic DNA Kit II 0913 Invisorb Genomic DNA Kit II Store diluted Proteinase K at 20 C but repeated freezing and thawing will reduced the activity dramatically Dividing the Proteinase K into aliquots and storage at 20 C is recommended 10 reactions 100 reactions 500 reactions Catalogue No 1032120100 1032120300 1032120400 Lysis Buffer G 3x2 ml 30 ml 160 ml Binding Buffer G 3x2 ml 60 ml 5 x 60 ml i for 250 ul for 2 ml for 5x 2 ml Proteinase K working solution working solution working solution Wash Buffer 2 ml 15 ml 4x 15 ml concentrate final volume 40 ml final volume 300 ml final volume 300 ml Elution Buffer 2x2ml 60 ml 200 ml Manual Initial steps Add 10 ml distilled water and 28 ml of 96 100 Ethanol to the Wash Buffer Concentrate then mix thoroughly add 250 ul ddH gt O to the Proteinase K Set a water bath ora Thermal Cycler or a Thermomixer to 50 C Lysis Buffer G 60 C Elution Buffer Add 75 ml distilled water and 210 ml of 96 100 Ethanol to the Wash Buffer Concentrate then mix thoroughly add 2 0 ml ddH 0 to the Proteinase K Set a water bath ora Thermal Cycler or a Thermomixer to 50 C Lysis Buffer G 60 C Elution Buffer Add 75 ml distilled water and 210 ml of 96 100 Ethanol to each Wash Buffer Concentrate then2. The length and delicate physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR Storage of DNA A working stock of DNA can be stored at 2 4 C for several weeks For long term storage DNA should be stored at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the solution in which the nucleic acid is eluted in will affect it s stability during storage Pure water lacks buffering capacity and an acidic pH may lead to acid hydrolysis Tris or Tris EDTA buffer contains sufficient buffering capacity to prevent acid hydrolysis Handling of DNA Avoid vigorous pipetting Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA DNA Yield The amount of purified DNA from the tissue or rodent tail sample depends on sample source transport conditions storage and age of the sample 13 Invisorb Genomi
3. conduct the prepared procedure Note Incubate Elution Buffer at 60 C Incubate Lysis Buffer at 50 C for 5 min Attention When using liver tissue please take not more than 20 mg 1 Place approximately 0 5 40 mg of fresh or frozen tissue material into a 1 5 or 2 0 ml reaction tube Note For tissue material which is difficult to be lysed for instance muscle or lung material we advise grinding the sample under liquid nitrogen A mechanical grinding of tissue with a glass rod can increase lysis efficiency too 2 Add 200 pl Lysis Buffer G and 20 ul Proteinase K for tissue samples up to 10 mg or 300 HI Lysis Buffer G and 20 HI Proteinase K for tissue samples gt 10 mg Incubation of the tube at 60 C continuous shaking for instance with a Thermomixer increases lysis process for lysis of starting material 3 Centrifuge for 1 min at 12 14 000 rpm for pelleting particels which are not lysed Transfer the supernatant into a new reaction tube 1 5 or 2 0 ml Note The Binding Buffer G has to be vortexed thoroughly before use 4 Addition of Binding Buffer G as described below Vortex the sample Incubation for 3 min at room temperature If the amount of tissue is 0 5 10 mg add 400 ul Binding Buffer if amount is gt 10 mg then add 600 ul 5 Centrifuge for 30 sec at 10 000 rpm short spin to pellet down the silica particles discard the supernatant carefully 6 Add 1 ml Wash Buffer then vortex briefly until the pellet is
4. mix thoroughly add 2 0 ml ddH20 to each Proteinase K Set a water bath or a Thermal Cycler or a Thermomixer to 50 C Lysis Buffer G 60 C Elution Buffer Invisorb Genomic DNA Kit II 0913 Symbols LoT Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation Do not reuse EO EKE F Manufacturer Storage All buffers and kit contents of the Invisorb Genomic DNA Kit ll except dissolved Proteinase K should be stored at room temperature RT and are stable for at least 12 months under these conditions Dissolved Proteinase K must be stored at 20 C Wash Buffer charged with ethanol should be stored at room temperature and should be appropriate sealed If there are any precipitates within the provided solutions dissolve these precipitates by carefully warming up to room temperature up to 30 C Room temperature RT is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb Genomic DNA Kit II for applications as described in this manual Purchaser must determine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecu
5. IANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of DNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used Product use limitation The kit is validated neither for the isolation of DNA from stool samples blood bacteria fungi or viruses nor for isolation and purification of RNA The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither implied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Pro
6. and discarded according to local safety regulations European Community risk and safety phrases for the components of the Invisorb Genomic DNA Kit II to which they apply are listed below as follows Binding Buffer G hb danger H225 H319 H336 P210 P233 P305 351 338 H319 Causes serious eye irritation H225 Highly flammable liquid and vapour H336 May cause drowsiness or dizziness P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P210 Keep away from heat sparks open flames hot surfaces No smoking P233 Keep container tightly closed Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 Reagents and equipment to be supplied by user Microcentrifuge Water bath set to 60 C Thermomixer or Thermal cycler Vortexer Microcentrifuge tubes 1 5 ml or 2 0 ml Distilled Water Milli Q 18M quality Ethanol 96 100 PBS Buffer TE Buffer Octane optional for protocol IIb oo O60 G 0O QO O 6 Invisorb Genomic DNA Kit II 0913 Sample collection and storage Tissue sample biopsy material frozen section Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this
7. ard supernatant and remove all media completely taking care not to disturb the cell pellet At this point cells may be frozen at 20 C or 80 C for future use or may be used immediately Cells grown in a monolayer Aspirate the media completely from the cells and continue immediately with the lysis step Alternatively cells can be detached by trypsination cultivation in larger culture vessels like dishes gt 5 35 mm flasks gt 12 5 cm Transfer cells to a 50 ml reaction tube pellet by centrifugation at 300 x g 1 500 rpm for 5 min and aspirate supernatant completely Buccal swabs To collect a sample scrape the swab firmly against inside of each cheek 6 times Air dry the swab for at least 2h after collection or use them fresh prepared Ensure that the person providing the sample has not consumed any food or drink in the 30 min prior to sample collection Best results are obtained if the swab stays in the lysis solution during lysis procedure Use of poor quality starting material influences yield of purified DNA This protocol is recommended for every common swab like e g the following swab types C E P Omni Swab from Whatman cotton swab Superswabs Copan Swab or DRACON tip from Hardwood Products company CellProjects or Hain Diagnostika CSF and Bone marrow on haematological slides Best results are obtained with fresh material But commonly the sample will be dried The have to be stored cooled at 4 C in a dried sur
8. c DNA Kit II 0913 Ordering information Product Package size Catalogue No Invisorb Genomic Kit II 10 preparations 1032120100 Invisorb Genomic Kit II 100 preparations 1032120300 Invisorb Genomic Kit II 500 preparations 1032120400 Single components for the Invisorb Genomic Kit II Lysis Buffer G 15 ml 1032121300 Binding Buffer G 15 ml 1032122000 Wash Buffer concentrate 15 ml 1032123000 Elution Buffer 15 ml 1032124000 Related products Invisorb Spin Tissue Mini Kit 50 preparations 1032100200 Invisorb Spin Tissue Mini Kit 250 preparations 1032100300 Invisorb DNA Tissue HTS 96 C 2 x 96 preparations 7032900200 Invisorb DNA Tissue HTS 96 C 4 x 96 preparations 7032900300 Invisorb DNA Tissue HTS 96 C 24 x 96 preparations 7032900400 14 Invisorb Genomic DNA Kit II 0913 E stratecee molecular STRATEC Molecular GmbH Robert R ssle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09 E mail info berlin stratec com www stratec com 1B3b 09 2013
9. completely resuspended then centrifuge at 10 000 rpm for 30 sec short spin Discard the supernatant carefully 7 Repeat the step 6 once again then briefly spin down the residual fluid and remove the residual Wash Buffer as completely as possible by pipetting Then incubate the open tube in a Vacuum dessicator or a water bath at 60 C for some minutes to completely evaporate the residual ethanol from the Wash Buffer Thermomixer or Thermal cycler can also be used 8 Addition of Elution Buffer prewarmed to 60 C Amount concerning to the table below or of Tris buffered ddH O pH 8 5 9 0 Thoroughly resuspend the pellet by pipetting up and down by manual vortexing Incubate at 60 C for 3 min 9 If the amount of tissue is 0 5 5 mg add 150 ul Elution Buffer if it is 5 10mg add 200ul Elution Buffer and from 10 40 mg add 300 400 ul Elution Buffer 10 Centrifuge at 14 000 rpm for 1 min and transfer the DNA containing supernatant into a new reaction tube Be very careful while pipetting the supernatant to avoid carry over of silica particles leave at least 2 ul supernatant above the pellet Note Regardless of how carefully the final DNA solution was separated from the Silica pellet some residual particles always remain in the solution which may affect the activity of DNA modifying enzymes To avoid associated problems we recommend centrifugation of the DNA solution for 2 minutes at 14 000 rpm each time prior to use A DNA aliquo
10. d only be used by trained personnel o O Oco O Preparing reagents and buffers Before starting a run bring all reagents to room temperature Where necessary gently mix and re dissolve any precipitates by incubation at 30 C Swirl gently to avoid foaming Lysis Buffer G and Elution Buffer are ready to use Add the needed volume of ddH O to the reaction tube with Proteinase K Vortex for 5 sec store diluted Proteinase K at 20 C 10 DNA extractions Add 10 ml distilled water and 28 ml of 96 100 Ethanol to the Wash Buffer Concentrate then mix thoroughly Add 250 ul ddH2O to the Proteinase K Set a water bath or a Thermal Cycler or a Thermomixer to 50 C Lysis Buffer G 60 C Elution Buffer 100 DNA extractions Add 75 ml distilled water and 210 ml of 96 100 Ethanol to the Wash Buffer Concentrate then mix thoroughly Add 2 0 ml ddH O to the Proteinase K Set a water bath or a Thermal Cycler or a Thermomixer to 50 C Lysis Buffer G 60 C Elution Buffer 500 DNA extractions Add 75 ml distilled water and 210 ml of 96 100 Ethanol to each Wash Buffer Concentrate then mix thoroughly Add 2 0 ml ddH20 to each Proteinase K Set a water bath or a Thermal Cycler or a Thermomixer to 50 C Lysis Buffer G 60 C Elution Buffer 8 Invisorb Genomic DNA Kit II 0913 Protocol I DNA isolation from fresh or frozen tissue samples 0 5 40mg Please read the instructions carefully and
11. d the supernatant carefully Repeat the step 7 once again then briefly spin down the residual fluid and remove the residual Wash Buffer as completely as possible by pipetting Then incubate the open tube in a Vacuum dessicator or a water bath at 60 C for some minutes to completely evaporate the residual ethanol from the Wash Buffer Thermomixer or Thermal cycler can also be used Note Strictly avoid overdrying of the Silica particles pellet Addition of 200 ul Elution Buffer or Tris buffered ddH20 pH 8 5 9 0 prewarmed to 60 C Thoroughly resuspend the pellet by pipetting up and down Incubate at 60 C for 3 min Centrifuge at 14 000 rpm for 1 min and transfer the DNA containing supernatant into a new reaction tube Be very careful while pipetting the supernatant to avoid carry over of silica particles leave at least 2 ul supernatant above the pellet Note Regardless of how carefully the final DNA solution was separated from the Silica pellet some residual particles always remain in the solution which may affect the activity of DNA modifying enzymes To avoid associated problems we recommend centrifugation of the DNA solution for 2 min at full speed each time prior to use A DNA aliquot for further use should be taken immediately after centrifugation from the top of the solution 10 Invisorb Genomic DNA Kit II 0913 Protocol III isolation of DNA from paraffin embedded tissue Please read the instructions carefully and conduc
12. ducts sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption 5 Invisorb Genomic DNA Kit II 0913 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF format at www stratec com for each STRATEC Molecular Product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the Invisorb Genomic DNA Kit II procedures for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered infectious and be handled
13. lar reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb Genomic DNA Kit II have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of Invisorb Genomic DNA Kit II or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor 4 Invisorb Genomic DNA Kit II 0913 Intended use The Invisorb Genomic DNA Kit II is the ideal tool for manual isolation and purification of genomic DNA from fresh or frozen human and animal tissues biopsy material rodent tail insects and paraffin embedded tissues as well as animal origin food samples and eucaryotic cells and cell pellets For reproducible and high yields appropriate sample storage is essential The purified DNA can be used for n vitro diagnostic analysis only THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY SUCH AS TECHNICIANS PHYSIC
14. leads to reduced DNA size Use of poor quality starting material influences yield of purified DNA The amount of purified DNA in the Invisorb Spin Tissue Mini Kit procedure using 5 40 mg tissue sample depends on kind of starting material The thawing process could proceed directly in Lyse Buffer G Rodent tail Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size A long time lysing also leads to degradation of DNA Crushing the rodent tails reduces lysis time Insects Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Insects especially with chitin mail must be homogenized before lysis for example by grinding with mortar and pestle under liquid nitrogen The sample can be stored for a short time at 2 8 C in Lyse Buffer G Paraffin slices formalin fixed tissue These samples can be stored at room temperature RT By appropriate paraffin embedding or formalin fixation pure DNA can be isolated from above named starting material but paraffin embedding or formalin fixation leads to reduced DNA quality An improper contact of the tissue with formalin will reduce dramatically the yield of DNA Cells grown in suspension Spin up to 1 x 10 cells for 5 min at 300 x g 1 500 rpm Disc
15. rom the top of the solution 11 Invisorb Genomic DNA Kit II 0913 Troubleshooting Problem probable cause Comments and suggestions low yield of extracted DNA ineffective destruction of starting material improper storage of starting material poor DNA binding by Silica particles substantial loss of silica particles bound DNA in the washing step due to low ethanol concentration in the Wash Buffer poor elution of the DNA bound on the Silica particles mechanical pre cutting of the material or grinding under liquid nitrogen increasing of lysis times store the samples carefully mix the sample with the Silica particles carefully prepare the Wash Buffer exactly as described in the manual storge of Wash Buffer with firmly fixed cap avoid over drying of the pellet prewarm Elution Buffer to 60 C bad results in PCR contamination of final DNA solution by chaotropic salts contamination of final DNA solution by particles of Silica particles contamination of final DNA solution by ethanol washing of the Silica particles pellet thoroughly as described in manual before using the DNA centrifuge the solution for 2min remove the ethanol carefully test the smell poor cleavage byrestriction endo nucleases or incomplete digestions of the purified genomic DNA see at bad PCR products 12 Invisorb Genomic DNA Kit II 0913 Appendix General notes on handling DNA Nature of DNA
16. rounding Note After Proteinase K digestion tissue samples can be stored in Lysis Buffer G for up to 6 month at 20 C without any reduction of DNA quality 7 Invisorb Genomic DNA Kit II 0913 Important notes Important points before starting a protocol Immediately upon receipt of the Product inspect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 7 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipette tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipette tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard gloves if they become contaminated Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed o This kit shoul
17. s stable long term storage at 4 C or 20 C Due to the high purity the isolated DNA is ready to use for a broad panel of downstream applications or can be stored at 20 C for subsequent use The kit is neither validated for the isolation of viral DNA or the purification of total RNA Trademarks Invisorb Registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invisorb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 Invisorb is a registered trademark of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2013 STRATEC Molecular all rights reserved 1 Invisorb Genomic DNA Kit II 0913 Contents Kit contents of Invisorb Genomic DNA Kit II Symbols Storage Quality control Intended use Product use limitation Safety information Reagents and equipment to be supplied by user Sample collection and storage Important notes Important points before starting a protocol Preparing reagents and buffers Protocol 1 DNA isolation from fresh or frozen tissue samples 0 5 25mg Protocol 2 Isolation of DNA from mouse tail Protocol 3 Isolation of DNA from paraffin embedded tissue Troubleshooting Appendix
18. stratecee molecular User manual Invisorb Genomic DNA Kit II For genomic DNA purification from 0 5 40 mg tissue sample mouse tail or paraffin embedded tissue batch system 1032120X00 dadli STRATEC Molecular GmbH D 13125 Berlin or Instructions of Invisorb Genomic DNA Kit II The Invisorb Genomic DNA Kit II has been developed for a fast and simple isolation of genomic DNA from tissue samples mouse tail and paraffin embedded tissue samples The kit completely avoids extractions with harmful and toxic organic solvents as well as ethanol precipitations and provides a set of convenient protocols for simultaneous analysis of multiple DNA samples per day In contrast to column based systems the Invisorb Genomic DNA Kit II batch system makes it possible to prepare genomic DNA even from smallest amounts of starting material and requires whether large amounts of starting material nor multiple phenol chloroform extractions Genomic DNA from large numbers of samples can be prepared very fast and with high recovery rates No special equipment is required The Invisorb Genomic DNA Kit II combines the cell and protein destructive properties of chaotropic compounds which result in the inactivation of endogenous DNases with binding of cellular DNA to silica particles Unique physical properties cause the excellent DNA binding capacity of the silica particles The excellent quality of the genomic DNA isolated with this kit allow
19. t for further use should be taken immediately after centrifugation from the top of the solution 9 Invisorb Genomic DNA Kit II 0913 Protocol Il isolation of DNA from mouse tail Please read the instructions carefully and conduct the prepared procedure Note Incubate Elution Buffer at 60 C Incubate Lysis Buffer at 50 C for 5 min 1 Cut about 0 8 cm of the mouse tail into small pieces and transfer the material into a 1 5 or 2 0 ml reaction tube If possible the material can also be grinded under liquid nitrogen so increasing the lysis efficiency Add 300 ul of Lysis Buffer G and 20 ul Proteinase K incubate at 60 C for lysis of the material continuous shaking for instance in a thermomixer increases lysis efficiency 4 Centrifuge for 1 min at 12 14 000 rpm for pelleting particels which are not lysed Transfer the supernatant into a new reaction tube 1 5 or 2 0 ml Note The Binding Buffer G has to be vortexed thoroughly prior to use 5 6 10 11 12 Addition of 600 ul Binding Buffer G and mix briefly and powerfully Incubate the reaction tube at room temperature for 3 min Centrifuge for 30 sec at 10 000 rpm short spin to pellet down the silica particles then discard the supernatant carefully Add 1 ml Wash Buffer resuspend the Silica particles pellet by pipetting up and down or by brief manual until the pellet is completely resuspended then centrifuge at 30 sec at 10 000 rpm short spin Discar
20. t the prepared procedure Note Incubate Elution Buffer at 60 C Incubate Lysis Buffer at 50 C for 5 min A Removing of paraffin 1 Place the starting material into a 1 5 ml reaction tube Add 1 ml Octan and vortex carefully to resolve the paraffin After resolving the paraffin the tissue sample becomes transparent while the paraffin is still white 2 Centrifuge at 14 000 rpm for pelleting the tissue sample and discard the supernatant carefully This step should be repeated if any paraffin is left in the sample 3 Add 0 5 ml 96 100 ethanol to the tissue sample and mix the tube thoroughly Short centrifugation and removing of the ethanol by aspiration with pipette Then incubate the open tube at 60 C for evaporating the residual ethanol B DNA Extraction 1 Add 200 ul of Lysis Buffer G and 20 ul Proteinase K incubate at 60 C for lysis of the material continuous shaking for instance in a thermomixer increases lysis efficiency 2 Transfer the whole cell lysis suspension into a 1 5 or 2 0 ml reaction tube Note The Binding Buffer G has to be vortexed thoroughly before use Addition of 400 ul Binding Buffer G and mix briefly and powerfully 4 Then incubate the reaction tube at room temperature for 3 min 5 Centrifuge for 30 sec at 10 000 rpm short spin to pellet down the silica particles then discard the supernatant carefully 6 Add imi Wash Buffer resuspend the Silica particles pellet by pipetting up and do
21. wn or by brief manual vortexing until the pellet is completely resuspended then centrifuge at 30 sec at 10 000 rpm short spin Discard the supernatant carefully 7 Repeat the step 7 once again then briefly spin down the residual fluid and remove the residual Wash Buffer as completely as possible by aspiration Then incubate the open tube in a Vacuum dessicator or a water bath at 60 C for some minutes to completely evaporate the residual ethanol from the Wash Buffer Thermomixer or Thermal cycler can also be used Note Strictly avoid overdrying of the Silica particles pellet 8 Addition of 100 ul Elution Buffer or Tris buffered ddH O pH 8 5 9 0 prewarmed to 60 C Thoroughly resuspend the pellet by pipetting up and down vortexing by manual vortexing 9 Incubation at 60 C for 3 min 10 Centrifuge at 14 000 rpm for 2 min and transfer the DNA containing supernatant into a new reaction tube Be very careful while pipetting the supernatant to avoid carry over of silica particles leave at least 2 ul supernatant above the pellet Note Regardless of how carefully the final DNA solution was separated from the Silica pellet some residual particles always remain in the solution which may affect the activity of DNA modifying enzymes To avoid associated problems we recommend centrifugation of the DNA solution for 2 min at full speed each time prior to use A DNA aliquot for further use should be taken immediately after centrifugation f
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