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Invisorb Spin Blood Midi Kit User manual

1. Midi Kit procedure 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 potentially infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the Invisorb Spin Blood Midi Kit to which they apply are listed below as follows Lysis Buffer A danger H319 P305 351 338 Proteinase K Wash Buffer I danger warning H315 319 334 335 P280 305 35 1 338 H302 312 332 412 EUH032 P273 H319 Causes serious eye irritation H315 Causes skin irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation H302 Harmful if swallowed H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas P305 P351 P338 IF IN EYES Rinse cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P280 Wear protective gloves protective clothing eye protection face protection P273 Avoid release to the environment 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 5 Invisorb Spin Blood M
2. ml 30 ml Buffer EL ready to use Buffer EL Concentrate 30 ml final volume 1000 ml Proteinase k Nea ee rena ae Wash Buteni a ee final Sea ml Wash Buffer Il a ane final ee ml Elution Buffer 2ml 15 ml Spin Filter 5 50 2 0 ml Receiver Tubes 10 2x 50 1 5 ml Receiver Tubes 5 50 Manual 1 1 Initial steps Add 21 ml 99 7 Isopropanol to the Add 250 ul ddH20 to the tube Proteinase K mix thoroughly and store at 20 C Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 30 ml of 96 100 ethanol to the bottle Wash Buffer I mix shortly and keep the bottle always firmly closed Add 42 ml of 96 100 Ethanol to the bottle Wash Buffer II mix shortly and keep the bottle always firmly closed Mix 30 ml Buffer EL Concentrate with 970 ml ddH20 mix very well and label the bottle with the Label Buffer EL Add 2 ml ddH20 to the tube Proteinase K mix thoroughly and store at 20 C Invisorb Spin Blood Midi Kit 0413 Symbols Manufacturer Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation o HKE AAE Do not reuse Storage All buffers and kit components of the Invisorb Spin Blood Midi Kit except dissolved Proteinase K should be stored at room temperature RT and are stable for 12 months under these conditions Room
3. of Erythrocytes and pelleting down of the nucleus containing cells Transfer of the blood sample into a 15 ml centrifuge tube and add 5 ml of cold Buffer EL 4 C mix shortly and incubate for 5 min Centrifuge at 2 250 x g 4 000 rpm for 5 minutes Carefully discard the supernatant Add again 5 ml of Buffer EL to the cell pellet and centrifuge at 3 500 x g 5 000 rpm for 5 minutes Carefully discard the supernatant If there is still a red color of the pellet please wash again with Buffer El Carefully discard the supernatant Important Don t remove the cell pellet Please note if you work with a fresh blood sample up to 4 hours after taking of the sample extend the lysis of erythrocytes by 30 minutes total time at least 45 min 2 Lysis at 70 C for 20 min in a thermo mixer Add 600 ul Lysis Buffer A to the cell pellet Pipett sometimes up and down and transfer the lysate completely into a 2 0 ml Receiver Tube Add 40 ul Proteinase K vortex shortly and incubate in a thermo mixer at 70 C for 20 min Place a Spin Filter into a 2 0 ml Receiver Tube 3 Realizing of optimal binding conditions Add 300 ul Binding Buffer A and vortex shortly 4 Loading the DNA binding Spin Filter Transfer 600 ul of the lysate onto the Spin Filter and incubate for 1 min Centrifuge at 11 000 x g 11 000 rpm for 2 min Discard the filtrate and place the Spin Filter back into the 2 0 ml Receiver Tube Reload the Spin Filter with the residual lysate and
4. 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 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 vacuum 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 DNA Yield The amount of purified DNA from the whole blood depends on the leucocytes content sample source transport storage and age Various different primary tubes and anticoagulants except heparin can be used to collect blood samples for the Invisorb procedure 14 Invisorb Spin Blood Midi Kit 0413 Ordering information Product Package size Catalogue No Invisorb Spin Blood Midi Kit 5 preparations 1031110100 Invisorb Spin Blood Midi Kit 50 preparations 1031110300 Single components for the Invisorb Spin Blood Midi Kit Lysis Buffer A 60 ml 1031111100 Binding Buffer A 30 ml 1031112100 Buffer EL concentrate 30 ml 1031115600 Wash Buffer add 30 ml 30 ml 1031113300 Wash Buffer II add 42 ml 18 ml 1031113400 Elution Buffer 15 ml
5. 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 Invisorb Spin Blood Midi Kit 0413 Contents Kit contents of the Invisorb Spin Blood Midi Kit Symbols Storage Quality control Intended use Product use limitation Safety information Product characteristic of the Invisorb Spin Blood Midi Kit Principle and procedure Sampling and storage of starting material Yield and quality of genomic DNA Important points before starting a protocol Preparing reagents and buffers of the Invisorb Spin Blood Midi Kit Reagents and equipment to be supplied by user O O DOAN NN OO OF FR FP WW W DY Important indications Scheme of the Invisorb Spin Blood Midi Kit Protocol 1 DNA extraction from 200 ul up to 1 ml blood a na N gt O Protocol 2 DNA extraction from 1 ml 2 ml blood 2 gt Troubleshooting A Appendix oi Ordering information 1 Invisorb Spin Blood Midi Kit 0413 Kit contents of the Invisorb Spin Blood Midi Kit Store dissolved Proteinase K at 20 C Store all other kit components at room temperature RT 5 DNA preps 50 DNA preps Catalogue No 1031110100 1031110300 Lysis Buffer A 2x2ml 60 ml ae 9 ml Binding Buffer A 2x1ml final volume 30
6. 1031114000 Related products Invisorb Spin Blood Mini Kit 50 preparations 1031100200 Invisorb Spin Blood Mini Kit 250 preparations 1031100300 Invisorb Blood Universal Kit 500 ml 1031150200 Invisorb Blood Universal Kit 1000 ml 1031150300 Invisorb Blood Mini 96 HTS C 4 x 96 preparations 7031300300 Invisorb Blood Mini 96 HTS C 24 x 96 preparations 7031300400 Invisorb Blood Mini 96 HTS R 4 x 96 preparations 7131300300 Invisorb Blood Mini 96 HTS R 24 x 96 preparations 7131300400 InviMag Blood DNA Mini Kit KFm 15 preparations 1031160100 InviMag Blood DNA Mini Kit KFm 75 preparations 1031160200 InviMag Blood DNA Mini Kit KF96 1 x 96 preparations 7431300100 InviMag Blood DNA Mini Kit KF96 5 x 96 preparations 7431300200 15 Invisorb Spin Blood Midi Kit 0413 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 1A3a02 04 2013
7. 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 samples are visible avoid aspirating them they could clog the Spin Filter membrane 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 Procedure Lysis of Erythrocytes and pelleting down the nucleus containing cells Lysis of the the nucleus containing cells Samples are lysed at elevated temperatures Lysis is performed in the presence of Lysis Buffer A and Proteinase K Binding genomic DNA By adding Binding Buffer A to the lysate optimal binding conditions will be adjusted Each lysate is then applied to a Spin Filter and genomic DNA is adsorbed to the membrane Removing residual contaminations Contaminants are efficiently washed away using Wash Buffers while the genomic DNA remains bound to the membrane Elution Genomic DNA is eluted from the Spin Filter using Elution Buffer The eluted DNA is ready for use in different downstream applications Eluted DNA stored at 4 8 C is stable for 2 months for more than 5 years if stored at 20 C Yield and quality of genomic DNA The amount of purified DNA using the Invisorb Spin Blood Midi Kit procedure depends on the sample type and the number of
8. Centrifuge for 2 min at 11 000 x g 11 000 rpm Discard the filtrate and place the Spin Filter back into the 2 0 ml Receiver Tube Add 500 ul Wash Buffer Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate and place the Spin Filter back into the 2 0 ml Receiver Tube Add 550 ul Wash Buffer Il Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard the filtrate place the filter again into the Receiver Tube repeat the step Discard the filtrate put the filter back into the 2 0 ml Receiver Tube then centrifuge for 4 min at maximum speed for ethanol removal Place the Spin Filter into a 1 5 ml Receiver Tube Add 100 ul Elution Buffer preheated to 70 C Incubate for 3 min at room temperature Centrifuge for 1 min at 11 000 x g 11 000 rpm Add again 100 ul of the Elution Buffer Centrifuge for 1 min at 11 000 x g 11 000 rpm Discard Spin Filter Close the 1 5 ml Receiver Tube and store the DNA sample at 4 C or 20 C 10 Invisorb Spin Blood Midi Kit 0413 Protocol 1 DNA extraction from 200 ul up to 1 mi blood Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 8 Important Transfer the needed amount of Elution Buffer into 2 0 ml Receiver Tube not included in the kit and place the tube at 70 C 1 Lysis of Erythrocytes and pelleting down the nucleus containing cells Transfer of 200 yl u
9. be thoroughly mixed and should have room temperature 3 The elution can be done by using lower amount of Elution Buffer This may result in a higher concentration of DNA But pay attention that minimum volume for elution is 50 ul but this will reduce the yield Elution volume between 2 x 50 ul up to 200 ul will realize comparable results 4 The eluted DNA volume will be lower than the added Elution Buffer volume Elution Buffer should be preheated to 70 C 5 The Elution Buffer doesn t contain EDTA 6 The yield can be increased if the incubation time with preheated Elution Buffer will be prolonged 7 Old blood samples often contains coagulates if coagulates or cryoprecipitate formed during thawing of frozen samples are visible avoid aspirating them they could clog the Spin Filter membrane 9 Invisorb Spin Blood Midi Kit 0413 Scheme of the Invisorb Spin Blood Midi Kit genomic DNA Please read protocols prior the start of the preparation carefully Transfer 200 ul up to max 1000 ul of blood into a 2 ml Receiver Tube add 1 ml of cold Buffer EL 4 C mix shortly and incubate for 5 min Centrifuge at 3 500 x g 5 000 rpm for 1 minute discard the supernatant Add 400 ul Lysis Buffer A and 40 pl Proteinase K vortex shortly Incubate for 20 min at 70 C while continuously shaking Add 200 ul Binding Buffer A follow preparing instructions and vortex shortly Transfer the solution onto Spin Filter incubate for 1 min
10. cells in the sample depending from the patients age and health situation sample source transport conditions storage and age of the sample Typically a 2 ml sample of whole blood cells from a healthy individual will yield up to 60 ug of DNA Samples with elevated white blood cell give a higher yield For most whole blood samples a single elution with 200 ul Elution Buffer is sufficient For samples with elevated white blood cell approximately 80 of the DNA will elute in the first 200 ul and up to 20 more in the next 200 ul Yield and quality of isolated genomic DNA is suitable for any molecular diagnostic detection system The diagnostic tests should be performed according to manufacturers specifications 7 Invisorb Spin Blood Midi Kit 0413 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 tips between liquid transfers To avoid cross contamination we recommend the use o
11. e 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 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 4 Invisorb Spin Blood Midi Kit 0413 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 Spin Blood
12. ed Spin insufficient lysis and or too increase lysis time Filter much starting material increase centrifugation speed or time reduce amount of starting material low amount of extracted DNA insufficient lysis incomplete elution insufficient mixing with Binding Buffer A increase lysis time reduce amount of starting material overloading of Spin Filter reduces yield prolong the incubation time with Elution Buffer to 5 10 min or repeat elution step once again take higher volume of Elution Buffer Mix sample with Binding Buffer A by pipetting or by vortexing prior to transfer the sample onto the Spin Filter Spin Filter surface turns yellow incomplete lysis insufficient washing too old starting material see above wash again with Wash Buffer II use protocol 2 low concentration of extracted DNA too much Elution Buffer elute the DNA with lower volume of Elution Buffer degraded or sheared DNA incorrect storage of starting material old material ensure that the starting material is fresh or stored under appropriate conditions for long time storage at 20 C avoid thawing and freezing of the material old material often contains degraded DNA genomic DNA does not perform well in downstream applications ethanol carryover during elution Salt carryover during elution Increase centrifugation time for of ethanol removal ensure that Wash Buffer is at room temperat
13. ep Add 550 ul Wash Buffer Il and centrifuge at 11 000 x g 11 000 rpm for 1min Discard the filtrate place the filter again into the Receiver Tube Repeat the washing step once again Discard the filtrate put the filter back into the 2 0 ml Receiver Tube and finally centrifuge for 4 min at maximum speed to remove the residual ethanol completely 7 Elution of the DNA Place the Spin Filter into a new 1 5 ml Receiver Tube and add 100 ul of preheated Elution Buffer Incubation for 3 min Centrifuge for 1 min at 11 000 x g 11 000 rpm Add again 100 ul of the Elution Buffer and centrifuge at 11 000 x g 11 000 rpm for 1 minute Note The DNA can also be eluted with a lower or a higher volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 50 ul If a quite large amount of DNA is expected the volume of elution can be increased 100 200 ul To maximize the final yield we recommend ever the use of two elution steps with an equal volume of Elution Buffer 11 Invisorb Spin Blood Midi Kit 0413 Protocol 2 DNA extraction from 1 ml 2 ml blood Please read the instructions carefully and conduct the prepared procedure Attention Please be aware that you have to prepare the Binding Buffer A see instruction page 8 Important Transfer the needed amount of Elution Buffer into 2 0 ml Receiver Tube not included in the kit and place the tube at 70 C 1 Lysis
14. f aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature o When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles o Discard gloves if they become contaminated o Do not combine components of different kits unless the lot numbers are identical o Avoid microbial contamination of the kit reagents o 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 should only be used by trained personnel Preparing reagents and buffers of the Invisorb Spin Blood Midi Kit Adjust the thermo mixer to 70 C Warm up the needed amount of Elution Buffer to 70 C 200 ul Elution Buffer are needed per sample Label the needed amount of Spin Filters Label the needed amount of 1 5 ml Receiver Tubes per sample 1 Receiver Tube Add the needed ul ddH20 to reaction tube with Proteinase K see below Vortex for 5 s Add the needed amount of ethanol to the Wash Buffers N ae o 5 DNA extractions add 250 ul ddH20 to Proteinase K mix thoroughly vortex 5s and store at 20 C Wash Buffers Binding Buffer A and Buffer EL are ready to use 50 DNA extractions add 21 ml 99 7 Isopropanol to the Binding Buffer A Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 2 ml ddH2O to Proteinase K m
15. idi Kit 0413 Product characteristic Starting material Yield Time for Invisorb Spin Blood Midi Kit preparation 200 ul up to 2 ml fresh up to 50 ug depends about 30 A260 A 280 whole blood samples on amount and specify minutes 1 6 2 0 frozen old or dry blood of starting material The Invisorb Spin Blood Midi Kit provides a very efficient procedure for isolation of high quality DNA directly from fresh frozen or old blood samples treated with citrate or EDTA The kit is designed for simultaneous processing of multiple samples After lysis of erythrocytes lymphocytes can be isolated free of any interfering hemoglobin The purification procedure of the kit is rapid and avoids the use of chaotropic components The DNA binds to filter membrane followed by washing steps and the final elution The purification procedure is rapid and requires neither phenol chloroform extraction nor alcohol precipitation 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 Due to the high purity the isolated genomic DNA is ready to use for a broad panel of downstream applications see below or can be stored at 20 C for subsequent use Downstream Application PCR real time PCR RFLP Analysis Restriction Enzyme Digestion Southern Blot Analysis Sequencing Cloning HLA typing STR Analysis SNP Analysis T
16. ix thoroughly vortex 5s and store at 20 C mix 30 ml Buffer EL Concentrate with 970 ml ddH20 mix very well and label the bottle with the Label Buffer EL add 30 ml of 96 100 ethanol to Wash Buffer I add 42 ml of 96 100 ethanol to Wash Buffer Il mix thoroughly and always keep the bottle firmly closed 8 Invisorb Spin Blood Midi Kit 0413 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 Microcentrifuge Thermomixer for 70 C Measuring cylinder 250 ml Disposable gloves Pipette and pipette tips Vortexer Reaction tubes 1 5 ml or 2 0 ml ddH 0 96 100 ethanol 1 x PBS optional Isopropanol oo co oc oC OC oO 8 The Invisorb Spin Blood Midi Kit is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol fur die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F Important indications 1 Process only as much blood samples as the micro centrifuge allows to process 2 Blood sample and buffers should
17. n Blood Midi 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 3 Invisorb Spin Blood Midi Kit 0413 Intended use The Invisorb Spin Blood Midi Kit is the ideal tool for a fast and convenient manual isolation and purification of genomic DNA from max 2 ml fresh frozen or old human blood For reproducible and high yields appropriate sample storage is essential The purified DNA can be used for in vitro diagnostic analysis only Fresh or frozen whole blood treated with EDTA or citrate but not with heparin from common blood collection systems can be used The protocols 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 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 conjunctio
18. n 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 on in vitro medical devices is not recognized Product use limitation The kit is neither validated for the isolation of genomic DNA from tissue serum plasma synovial fluid and urine nor from bacteria stool sample fungi parasites or the purification of total RNA The application of the kits for isolation and purification of viral DNA has not been evaluated The kits applicability for cultured or isolated cells tissue stool sample swabs dried blood stains or cell free body fluids like cerebrospinal fluid synovial fluid and urine also have not been validated 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 b
19. o purify genomic DNA in 96 format STRATEC Molecular offers the Invisorb Blood Mini HTS 96 Kit for use in a centrifuge and on common laboratory automated workstations Furthermore STRATEC Molecular offers the InviMag Blood Midi Kits for DNA isolation using magnetic beads For minimal amount of starting material the Invisorb Spin Blood Mini Kit max 200 ul is available To purify genomic DNA from large volumes of blood STRATEC Molecular offers the Invisorb Blood Universal Kit 1 10 ml For further information please contact Tel 49 0 30 9489 2901 2910 in Germany and from foreign countries Tel 49 0 30 9489 2907 or your local distributor O Or O O Or 0r OOO The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 6 Invisorb Spin Blood Midi Kit 0413 Principle and procedure The Invisorb Spin Blood Midi Kit procedure comprise following steps lysis of erythrocytes lysis of nucleus containing cells binding the genomic DNA to the membrane of Spin Filter washing the membrane and elimination of ethanol elution of genomic DNA OE Oe This manual contains 2 protocols according to the different requirements of the starting materials Sampling and storage of starting material Blood Blood samples stabilized with EDTA or Citrate can be stored at room temperature for 2 3 hours for short time storage up to 24 h samples may be stored at 4
20. p to max 1 ml of the blood sample into a 2 0 ml reaction tube and add 1 ml of cold Buffer EL 4 C mix shortly and incubate for 5 min Centrifuge at 3 500 x g 5 000 rpm for 1 minute RT Carefully discard the supernatant If the lymphocyte pellet have a red color please add 1 ml of Buffer EL vortex shortly and centrifuge at 3 500 x g 5 000 rpm for 1 minute again If there is still a red color of the pellet please wash again with Buffer El Carefully discard the supernatant Important Don t remove the cell pellet Please note if you work with a fresh blood sample up to 4 hours after taking of the sample extend the lysis of erythrocytes by 30 minutes total time at least 45 min 2 Lysis at 70 C for 20 min in a Thermo mixer Add 400 ul Lysis Buffer A and 40 ul Proteinase K vortex shortly and incubate in a shaking Thermo mixer at 70 C for 20 min Place a Spin Filter into a 2 0 ml Receiver Tube 3 Realizing of optimal binding conditions Add 200 ul Binding Buffer A and vortex shortly 4 Loading the DNA binding Spin Filter Transfer the solution onto the Spin Filter and incubate for 1 min Centrifuge at 11 000 x g 11 000 rpm table centrifuge for 2 min Discard the filtrate and place the Spin Filter back into the 2 0 ml Receiver Tube 5 First Washing step Add 500 ul Wash Buffer and centrifuge at 11 000 x g 11 000 rpm for 1min Discard the filtrate place the filter back into the 2 0 ml Receiver Tube 6 Second Washing st
21. repeat the centrifugation step Discard the filtrate and place the Spin Filter back into the 2 0 ml Receiver Tube 5 First Washing step Add 500 ul Wash Buffer and centrifuge at 11 000 x g 11 000 rpm for 1min Discard the filtrate place the filter back into the 2 0 ml Receiver Tube 6 Second Washing step Add 550 ul Wash Buffer Il and centrifuge at 11 000 x g 11 000 rpm for 1min Discard the filtrate place the filter again into the Receiver Tube Repeat the washing step once again Discard the filtrate put the filter back into the 2 0 ml Receiver Tube and finally centrifuge for 4 min at maximum speed to remove residual ethanol completely 7 Elution of the DNA Place the Spin Filter into a new 1 5 ml Receiver Tube and add 100 ul of preheated Elution Buffer Incubation for 3 min Centrifuge for 1 min at 11 000 x g 11 000 rpm Add again 100 ul of the Elution Buffer and centrifuge at 11 000 x g 11 000 rpm for 1 minute Note The DNA can also be eluted with a lower or a higher volume of Elution Buffer depends on the expected yield of genomic DNA But pay attention that the minimum volume for the elution is 50 pl If a quite large amount of DNA is expected the volume of elution can be increased 100 200 pl To maximize the final yield we recommend ever the use of two elution steps with an equal volume of Elution Buffer 12 Invisorb Spin Blood Midi Kit 0413 Troubleshooting Problem Cause Comments and suggestions clogg
22. stratecee molecular User manual Invisorb Spin Blood Midi Kit For purification of genomic DNA from 200 2000 ul fresh or frozen whole mammalian blood with the common anticoagulants EDTA Citrate 1031110X00 nell STRATEC Molecular GmbH D 13125 Berlin or Instruction for the Invisorb Spin Blood Midi Kit The Invisorb Spin Blood Midi Kit is the ideal tools using the Invisorb technology for a fast efficient and simple manual isolation and purification of genomic DNA f from up to 2 ml of fresh frozen with common anticoagulants EDTA Citrate blood The purified DNA can be used for in vitro diagnostic analysis The kit is neither validated for the isolation of genomic DNA from tissue serum plasma synovial fluid and urine nor from bacteria stool sample 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 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
23. 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 Wash Buffer charged with ethanol should be appropriately sealed and stored at room temperature 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 Spin Blood Midi 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 ISO EN 13485 certified Quality Management System the performance of all components of the Invisorb Spin Blood Midi 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 Spi
24. ure check up Wash Buffer for salt precipitates If there are any precipitates dissolve these precipitates by careful warming 13 Invisorb Spin Blood Midi Kit 0413 Appendix General notes on handling DNA 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 lyses conditions of blood differ depending on the 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 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 and 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

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