Invisorb Blood Universal Kit User manual - Negev Bio
Contents
1. 20 ml For minimal amount of starting material the Invisorb Spin Micro DNA Kit max 50 ul is available For blood stains STRATEC Molecular offers the Invisorb Forensic Kit I To purify genomic DNA in 96 format STRATEC Molecular offers the Invisorb Blood Mini HTS 96 Kit for use in a centrifuge on a vacuum manifold and on common workstations Furthermore STRATEC Molecular offers the InviMag Blood Mini Kits for DNA isolation using magnetic beads For technical support or further information please contact 49 0 30 9489 2907 2910 or your local distributor The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG T Invisorb Blood Universal Kit 0213 Principle and procedure The Invisorb Blood Universal Kit procedure comprises following steps Removal of erythrocytes Lysis of Lymphocytes Precipitation of DNA leaving behind contaminants in the supernatant Dissolution of genomic DNA ie oe Oo Sampling and storage of starting material Blood and Buffy Coat Mammalian blood samples stabilized with EDTA or Citrate can be stored at room temperature 18 25 C for 2 3 hours for short time storage up to 24 h samples may be stored at 4 C For long term storage we recommend freezing samples at 20 C or 80 C Multiple thawing and freezing before isolating the DNA should be avoided If cryoprecipitate formed during thawing of frozen sam2. Yield and quality of genomic DNA The amount of purified DNA in the Invisorb Blood Universal Kit procedure from whole blood depends on the leucocytes content sample source transport storage and age of the sample Even human blood is different between individuals depending on age health and conditions of life If you are using blood from animals keep in mind that lysis conditions of blood differs depending on species Also remember that non mammalian blood contains erythrocytes with nuclei So for special applications adaptation of starting volumes and lysis time may be recommended Yield and quality of isolated genomic DNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed according to manufacturer s specifications 8 Invisorb Blood Universal Kit 0213 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 6 Do not use damaged kit components since their use may lead to poor kit performance O Always change pipet ti
3. Blood Universal Kit 0213 Symbols Manufacturer Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation Do not reuse HKL A aE Storage All buffers and kit components of the Invisorb Blood Universal Kit except dissolved Proteinase K should be stored at room temperature RT and are stable for 12 months under these conditions Room temperature RT is defined as range from 15 30 C Dissolved Proteinase K stored at 20 C is stable for 12 months but repeated freezing and thawing should be avoided Aliquotation and storage at 20 C is recommended If there are any precipitates within the provided solutions dissolve these precipitates by carefully warming up to room temperature up to 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb Blood Universal Kit 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 Molecular 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
4. 9 0 or b 10 mM Tris HCI pH 8 5 9 0 2 Process only as much blood samples as the centrifuge allows to process 3 Itis recommended to mix the isolated DNA after long storage longer than 24 h before use in any application 4 Blood sample and Buffers should be thoroughly mixed and should have room temperature 18 25 C 5 The Buffer EL has to be cooled down to 4 C see protocol 2 before use Attention If you decant Buffer El from pelleted lymphocytes be sure that you don t detach the pellet depending on the blood it can be not very well attached The composition of plasma and lymphocytes in blood differs depending on age and health status of the patient 10 Invisorb Blood Universal Kit 0213 Scheme of the Invisorb Blood Universal Kit Please read protocols prior the start of the preparation carefully Add needed amount of cold Buffer EL 4 C to 15 or 50 ml Reaction Tube Add whole blood sample Mix thoroughly and incubate on ice for 10 min Centrifuge at 1000 x g 3 000 rpm for 5 min Remove the supernatant carefully see page 10 Wash again with Buffer EL vortex shortly Centrifuge again at 1 000 x g 3 000 rpm for 5 min Remove the supernatant very carefully invert the tube and dab it on a paper tissue to remove the residual fluid Add the needed amount of Lysis Buffer HL and Proteinase K depending on the starting volume of whole blood sample to the cell pellet Incubate the tube at 60 C for 1
5. other commercially available methods The simple purification procedure is performed without spin filter in a single tube which reduces costs and simplifies handling The kit uses a special lysis reagent for selective lysis of erythrocytes After all erythrocyte components are removed leukocyte DNA can be isolated free of interfering hemoglobin The remaining leukocytes are lysed with optimized buffer and proteins are removed by protein digestion DNA is precipitated selectively by addition of Precipitation Solution recovered by centrifugation washed and dried DNA is resuspended in elution buffer and is ready for direct use in downstream assays or for storage at 20 C The concentration of the resulting DNA can be adapted to the needs of the user The isolated DNA is highly chromosomal and well suitable for archiving Downstream Applications PCR expand long template PCR HLA typing Restriction Enzyme Digestion Cloning Southern Blot oO000 0 The procedure requires no phenol chloroform extraction and requires minimal interaction by the user allowing safe handling of potentially infectious samples The procedure is designed to avoid sample to sample cross contamination To purify genomic DNA from other volumes of whole blood STRATEC Molecular offers the Invisorb Spin Blood Mini Kit max 200ul the Invisorb Spin Blood Midi Kit max 2 ml the Invisorb Spin Blood Maxi Kit max 10 ml and the Invisorb Blood Giga Kit 0 2
6. 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 Blood Universal Kit 0213 Contents Kit contents of the Invisorb Blood Universal Kit 3 Symbols 4 Storage 4 Quality control 4 Intended use 5 Product use limitation 5 Safety information 6 Product characteristic of the Invisorb Blood Universal Kit 7 Principle and procedure 8 Sampling and storage of starting material 8 Yield and quality of genomic DNA 8 Important notes 9 Important points before starting a protocol 9 Preparing reagents and buffers 9 Reagents and equipment to be supplied by user 10 Important indications 10 Scheme 11 Protocol 1 DNA isolation from 1 10 ml human and mammalian whole blood or from the corresponding amount of buffy coat 12 Troubleshooting 14 Appendix 15 Ordering information 16 2 Invisorb Blood Universal Kit 0213 Kit contents of the Invisorb Blood Universal Kit Store dissolved Proteinase K at 20 C Store all other kit components at room temp
7. 0 405 H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up 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 6 Invisorb Blood Universal Kit 0213 Product characteristic of the Invisorb Blood Universal Kit up to 10 ml fresh frozen or up to 400 ug depends on approx 45 min A260 A 280 old whole blood EDTA amount of lymphocytes 1 7 1 9 citrate corresponding sample source transport amount of buffy coat storage and sample age The Invisorb Blood Universal Kit provides an alternative approach for the isolation of DNA from 1 0 10 ml mammalian whole blood etc Traditionally preparing such DNA from blood requires removal of haemoglobin by labor intensive methods such as density gradients and removal of proteins and lipids with hazardous solvents like phenol chloroform Sambook et al 1989 The procedure of the Invisorb Blood Universal Kit is faster to perform than homemade or
8. 0 min Prefill the described amount of Precipitation Solution into 15 ml tubes and add the lysate to the prefilled tubes Mix the tube vigorously by inverting Centrifuge at 2 000 x g 4 500 rpm for 5 min Remove the supernatant invert the tube and dab it on paper tissue Add needed amount of 70 Ethanol to the DNA pellet Vortex shortly Centrifuge at 2 000 x g 4 500 rpm for 5 min Remove the supernatant invert the tube and dab it on paper tissue Remove ethanol by pipetting or drying Add the needed amount of Buffer U to the pellet Vortex shortly Incubate at 60 C for at least 1 h genomic DNA 11 Invisorb Blood Universal Kit 0213 Protocol Isolation of genomic DNA from whole blood up to 10 ml or from buffy coat corresponding amount Please read the instructions carefully and conduct the prepared procedure Note If buffy coat should be used start directly with the cell lysis step Lysis of Erythrocytes 1 Prefill the described amount of Buffer EL 4 C into 15 ml or 50 ml Reaction Tubes and add the whole blood sample to the Buffer EL see table Volume of whole blood ml 1 2 3 4 5 6 7 8 9 10 Buffer EL ml 2 5 5 0 7 5 10 0 12 5 15 0 17 5 20 0 22 5 25 0 Reaction Tube ml 15 15 15 50 50 50 50 50 50 50 2 Mix the sample with the Buffer EL very well and place the tube on ice for 10 min Note If buffy coat is used for the calculations of vol
9. For reproducible and high yields appropriate sample storage is essential The protocol for the isolation and all buffers are optimized for a high yield as well as a high purity All hands on steps are reduced to a minimum This kit is designed for extraction of DNA from blood but even human blood is different between individuals depending on age health and conditions of life If you are using blood from animals keep in mind that lyses conditions of blood differs depending on species Also remember that non mammalian blood contains erythrocytes with nuclei So for special applications adaptation of starting volumes and lyses time may be recommended THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY SUCH AS TECHNICIANS PHYSICIANS 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 The kit is in compliance with EU Directive 98 79 EC on in vitro medical devices But it is not for in vitro diagnostic use in countries where the EU Directive 98 79 EC o
10. ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb Blood Universal Kit 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 Blood Universal Kit 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 Blood Universal Kit 0213 Intended use The Invisorb Blood Universal Kit provides a very fast efficient and careful procedure for the isolation of high quality DNA from 1 10 ml of whole blood or from corresponding amount of buffy coat The procedure can be used for native blood as well as for blood treated with EDTA or citrate but not with heparin from common blood collection systems The concentration of the final DNA can be adapted to the individual needs of the user for instance very highly concentrated DNA for HLA typing The high purity of the genomic DNA guarantees storage stability at 4 C or at 20 C for several months without degradation Hence it enables the possibility to ship the DNA without any problems
11. al applications adaptation of starting volumes and lysis time may be recommended The procedure can be used for native blood as well as for blood treated with EDTA or citrate but not with heparin from common blood collection systems 2 Nature of DNA 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 3 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 4 Drying dissolving and pipetting DNA Avoid over drying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum although va
12. cuum drying can be used with caution 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 5 DNA yield The amount of purified DNA from the whole blood depends on the leucocytes content sample source human animal age of the individuals health situation transport storage and age of the sample Various different primary tubes and anticoagulants except heparin can be used to collect blood samples for the Invisorb procedure 15 Invisorb Blood Universal Kit 0213 Ordering information Product Package size Catalogue No Invisorb Blood Universal Kit 50 ml preparation 1031150100 Invisorb Blood Universal Kit 500 ml preparation 1031150200 Invisorb Blood Universal Kit 1000 ml preparation 1031150300 Invisorb Spin Blood Mini Kit 50 preparations 1031100200 Invisorb Spin Blood Mini Kit 250 preparations 1031100300 Invisorb Spin Blood Maxi Kit 50 preparations 1031120300 Invisorb Blood Mini HTS 96 Kit C 4 x 96 preparations 7031300300 Invisorb Blood Mini HTS 96 Kit C 24 x 96 preparations 7031300400 Invisorb Blood Mini HTS 96 Kit R 4 x 96 preparations 7131300300 Invisorb Blood Mini HTS 96 Kit R 24 x 96 preparations 7131300400 InviMag Blood DNA Mini Kit KFml 15 preparations 2431110100 InviMag Blood DNA Mini Kit KFml 75 preparations 2431110200 Inv
13. d or the centrifugation speed can be increased Drying of the DNA Pellet 13 From small amounts of blood up to 2 ml Dry the DNA pellet on the air until all traces of ethanol have evaporated The time for drying should be short as possible about 5 min From bigger amounts of blood gt than 2 ml Spin down again residual ethanol and remove completely the supernatant with a small volume pipette Attention please Over dried DNA is very difficult to dissolve The DNA isolated with this kit is of high quality and in big amounts of high complexity so the dissolving process takes some time Dissolving of the DNA 14 Add the described amount of Buffer U to the pellet see table Volume of whole blood ml 1 2 3 4 5 6 7 8 9 10 Buffer U ml 0 2 04 06 08 10 12 14 16 1 8 2 0 15 Vortex shortly max 3 5 sec Dissolve the DNA completely by incubation at 50 C for at least 1h The DNA can be also dissolved at room temperature overnight Continuously shaking or inverting of the tube from time to time min 5 x increases the dissolving efficiency Optional transfer the DNA into a 1 5 ml or 2 0 ml Reaction Tube 13 Invisorb Blood Universal Kit 0213 Troubleshooting Problem Cause Comments and suggestions low amount of DNA insufficient lysis incompletely dissolved DNA low DNA concentration in the sample increase lysis time with Buffer EL make su
14. erature RT for preparation from 50x 1 ml for preparation from 500 x 1 ml for preparation from 1000 x 1 ml Proteinase K working solution working solution up to 5 x 10 ml up to 50 x 10 ml up to 100 x 10 ml Catalogue No 1031150100 1031150200 1031150300 250 ml 3x 30m 6x 30m Buffer EL ready to use final volume 1000 mi final volume 1000 ml Lysis Buffer HL 30 ml 2 x 140 ml 2 x 270 ml for 300 ul for 3 x 1 ml for 5 x 1 1 ml working solution Precipitation Solution fill with 99 7 Isopropanol empty bottle final volume 30 ml 2 empty bottles final volume 2 x 140 ml 2 empty bottles final volume 2 x 270 ml isopropanol molecular biologic grade into the empty bottle Add 300 ul ddH20 to Proteinase K mix thoroughly and store at 20 C Isopropanol molecular biologic grade into each empty bottle Add 1 ml ddH 0 to Proteinase K mix thoroughly and store at 20 C Mix thoroughly 970 ml of ddH 0 with the 30 ml Buffer EL concentrate and keep the bottle always firmly closed Buffer U 15 ml 2 x60 ml 250 ml Manuals 1 1 1 Initial steps Fill 30 ml 99 7 Fill 140 ml 99 7 Fill 270 ml 99 7 isopropanol molecular biologic grade into each empty bottle Add 1 1 ml ddH 0 to Proteinase K mix thoroughly and store at 20 C Mix thoroughly 970 ml of ddH 0 with the 30 ml Buffer EL concentrate and keep the bottle always firmly closed Invisorb
15. iMag Blood DNA Mini Kit KF96 1 x 96 preparations 7431300100 InviMag Blood DNA Mini Kit KF96 5 x 96 preparations 7431300200 Possible suppliers for lsopropanol Fa Carl Roth 2 Propanol 7 Fa Applichem Fa Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol f r die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F 16 Invisorb Blood Universal Kit 0213 Stratecee molecular STRATEC Molecular GmbH Robert Rossle 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 1B3a 02 2013
16. mproves lysis efficiency If you don t use a thermomixer small sample volume it is recommended to mix the sample three time during incubation 12 Invisorb Blood Universal Kit 0213 DNA Precipitation 7 Prefill the described amount of Precipitation Solution into 15 ml tubes and add the lysate to the prefilled tubes see table Volume of whole blood ml 1 2 3 4 5 6 7 8 9 10 Precipitation Solution ml 0 5 1 0 1 5 20 25 30 35 40 4 5 5 0 8 Mix the tube vigorously by inverting until the DNA flakes becomes visible 9 Centrifuge at 2 000 x g 4 500 rpm for 5 min Remove the supernatant very carefully invert the tube and dab it on paper tissue to remove the residual fluid Be carefully Don t decant the pellet If the DNA pellets are very loose centrifugation can be prolonged or the centrifugation speed can be increased Washing of the DNA Pellet 10 Add the described amount of 70 Ethanol to the DNA pellet see table Volume of whole blood ml 1 2 3 4 5 6 7 8 9 10 Ethanol 70 ml 0 5 1 0 15 20 25 30 35 40 45 5 0 11 Vortex shortly 12 Centrifuge at 2 000 x g 4 500 rpm for 5 min Remove the supernatant very carefully invert the tube and dab it on paper tissue to remove the residual fluid Be carefully Don t decant the pellet If the DNA pellets are very loose centrifugation can be prolonge
17. n in vitro medical devices is not recognized Product use limitation The kit is neither validated for the isolation of genomic DNA from cultured or isolated cells from tissue stool sample swabs dried blood stains or cell free body fluids like cerebrospinal fluid synovial fluid and urine nor from bacteria fungi parasites or the purification of RNA The application of the kit for isolation and purification of viral DNA has not been evaluated 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 Products 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 detecti
18. on 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 Blood Universal Kit 0213 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 Blood Universal Kit 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 and discarded according to local safety regulations European Community risk and safety phrases for the components of the Invisorb Blood Universal Kit to which they apply are listed below as follows Proteinase K OS danger H315 319 334 335 P280 305 351 338 3 1
19. ples are visible avoid aspirating them Various different primary tubes blood collection system e g Sarstedt Greiner and anticoagulants except heparin can be used to collect blood samples for the Invisorb procedure Buffy coat is a whole blood fraction of enriched leukocyte cells To prepare and extract a buffy coat layer the following procedure is recommended The use of a whole blood sample anticoagulants EDTA citrate not heparin with a sedimented cellular fraction from staying overnight at 4 C is recommended The resulting bright mid section overlaid by the clear plasma is buffy coat containing concentrated leukocytes that can be easily distinguished from the erythrocytes in the bottom layer An enrichment factor of 10 is expected from such a procedure Due to the enriched leukocyte content be aware to avoid overloading the DNA purification procedure Lysis Samples are lysed at elevated temperature Lysis is performed in the presence of Lysis Buffer HL and Proteinase K Precipitation of DNA By adding Precipitation Solution to the lysate optimal precipitation conditions will be adjusted DNA is separated by centrifugation contaminants remain in the supernatant Suspending DNA Genomic DNA is dissolved using 400 2000 ul Buffer U The dissolved eluted DNA is ready for use in different downstream applications Eluted DNA stored at 4 8 C is stable for a minimum of 2 months or is stable for more than 5 years stored at 20 C
20. propanol 99 7 molecular biologic grade into each empty bottle Add 1 1 ml ddH O to the tube with Proteinase K Mix thoroughly vortex 5 sec and store at 20 C Mix thoroughly 970 ml of ddH O with 30ml Buffer EL concentrate and always keep the bottle firmly closed 9 Invisorb Blood Universal Kit 0213 Reagents and equipment to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles 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 under each STRATEC Molecular kit and kit component Centrifuge Thermomixer for 60 C Ice Measuring cylinder 250 ml Disposable gloves Pipette and pipette tips Vortexer dd H20 70 ethanol Isopropanol The Invisorb Blood Universal Kit is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth oO OO 0 60 G oOo 6 Q Possible suppliers for lsopropanol Sealer Fa Applichem Fa Sigma ee 2 Propanol f r die Molekularbiologie 2 Propanol Rotipuran 799 7 p a ACS ISO Qrder no A3928 Order no 59304 1L F Order no 6752 Important indications 1 The DNA can be concentrated by using lower amounts of Buffer U Be sure that you dissolve the pellet completely We recommend the following buffers to dilute the DNA a 10 mM Tris HCI 0 1mM EDTA pH 8 5
21. ps between liquid transfers To avoid cross contamination the use of aerosol barrier pipet tips is recommended 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 This kit should only be used by trained personnel Preparing reagents and buffers f 2 adjusting the thermomixer to 60 C add the needed ul ddH20O to Proteinase K and Buffer EL see below Vortex for 5 sec for DNA extraction from 50 ml blood Fill 30 ml 99 7 Isopropanol molecular biologic grade into the empty bottle Add 300 ul ddH20 to the tube with Proteinase K Mix thoroughly vortex 5 sec and store at 20 C Buffer EL is ready to use for DNA extraction from 500 ml blood Fill 140 ml 99 7 Isopropanol molecular biologic grade into each empty bottle Add 1 ml ddH20 to the tube with Proteinase K Mix thoroughly vortex 5 sec and store at 20 C Mix thoroughly 970 ml of ddH20 with 30 ml Buffer EL concentrate and always keep the bottle firmly closed for DNA extraction from 1000 ml blood Fill 270 ml iso
22. re that the Buffer EL is cooled down to 4 C increase lysis time with Lysis Buffer HL continuously shaking improves lysis efficiency increase incubation time with Buffer U dilute the DNA with lower volume of Buffer U diluted DNA tints yellow insufficient lysis of erythrocytes inefficient washing increase lysis time with Buffer EL make sure that the Buffer EL is cooled down to 4 C wash the lymphocyte pellet again with Buffer EL wash again with 70 ethanol degraded or sheared DNA incorrect storage of starting material old material ensure the sample is harvested and stored as described avoid repeated thawing and freezing of the material old material often contains degraded DNA problems with subsequent applications e g in PCR ethanol in the eluated DNA salt in the eluat spin down the DNA and remove all traces of ethanol by pipetting increase the time for the elimination of ethanol Check correct calculation of volumes Invisorb Blood Universal Kit 0213 Appendix General notes on handling DNA 1 Starting material This kit is designed for extraction of DNA from blood but even human blood is different between individuals depending on age health and conditions of life If you are using blood from animals keep in mind that lysis conditions of blood differs depending on the species Also remember that non mammalian blood contains erythrocytes with nuclei So for speci
23. stratecee molecular User manual Invisorb Blood Universal Kit For purification of genomic DNA from 1 10 ml of whole mammalian blood with the common anticoagulants EDTA citrate 1031150x00 easi STRATEC Molecular GmbH D 13125 Berlin Er Instruction for the Invisorb Blood Universal Kit The Invisorb Blood Universal Kit provides a very fast efficient and careful procedure for the isolation of high quality DNA from 1 10 ml of whole blood or from buffy coat The procedure can be used for native blood as well as for blood treated with common anticoagulants EDTA citrate The concentration of the final DNA can be adapted to the individual needs of the user for instance very highly concentrated DNA for HLA typing The purified DNA can be used for in vitro diagnostic analysis The kit is neither validated for the isolation of genomic DNA from cultured or isolated cells from tissue swabs dried blood stains or cell free body fluids like cerebrospinal fluid synovial fluid and urine stool sample nor from bacteria fungi parasites or the purification of total RNA The application of the kit for isolation and purification of viral DNA has not been evaluated Compliance with EU Directive 98 79 EC on in vitro medical devices Not for in vitro diagnostic use in countries where the EU Directive 98 79 EC on in vitro medical devices is not recognized Trademarks Invisorb Registered marks trademarks etc
24. umes in the tables take the volume of blood from which you made the buffy coat If you have really clean buffy coat you may start with step 3 from this protocol If you are not sure that erythrocytes are removed quantitatively it is recommended to do a treatment with buffer EL starting with step two Collecting of nucleated blood cells 3 Centrifuge at 1 000 x g 3 000 rpm for 5 min Remove the supernatant carefully and wash again with the same volume Buffer EL as before vortex shortly 4 Centrifuge again at 1 000 x g 3 000 rpm for 5 min Remove the supernatant very carefully invert the tube and dab it on a paper tissue to remove the residual fluid the lymphocytes should be visible at the ground of the tube Be careful don t decant the cell pellet Cell Lysis 5 Add the described amount of Lysis Buffer HL and Proteinase K depending on the starting volume of whole blood sample to the cell pellet see table Volume of whole blood ml 1 2 3 4 5 6 7 8 9 10 Lysis Buffer HL ml 05 1 0 1 5 20 25 30 35 40 45 5 0 Proteinase K pl 5 10 15 20 25 30 35 40 45 50 Important Note When processing multiple blood sample vortex each tube immediately after addition of Lysis Buffer HL and Proteinase K Do not wait until the Lysis Buffer HL and Proteinase K had been added to all samples 6 Incubate the tube at 60 C for 10 min in a water bath or heating block etc Agitation i
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