MagJET Whole Blood gDNA Kit
Contents
1. wells of row A of the Blood DNA plate 1 Add the following reagents to the rows Note that row B of the Blood DNA plate 1 is reserved for the tip and should be left empty Note that rows C of the Blood DNA plate 1 and D of the Blood DNA plate 2 are left empty too Plate name and type Row Row name Content Sample reagent volume per well Blood sample 1000 uL seats A Sample Lysis Buffer 500 uL KingFisher Ley deep well l UE BH plate B Tip 12 tip comb Empty C Empty Empty Empty D Wash 1_1 Wash Buffer 1 3500 uL EE E A Elution Elution Buffer 900 uL AA B Wash 2 Wash Buffer 2 5000 uL a errex t deep wel o T Washt 2 Wash Buffert 3500 uL D Empty Empty Empty 3 Place a Thermo Scientific KingFisher Duo 6 tip comb into row B of the Blood DNA plate 1 4 Switch on the KingFisher Duo and make sure that you are using the KingFisher Duo 6 pin magnet head and heating block Start the Blood_gDNA_1mL_Duo protocol Insert the Blood DNA plates into the instrument as indicated on the KingFisher Duo display After both plates have been loaded into the instrument the protocol will begin 5 Add the MagJET Magnetic beads resuspended well by vortexing and Binding Buffer supplemented with ethanol see page 5 to row A of the Blood DNA plate 1 when the KingFisher Duo pauses at the dispense step after the lysis step approximately 10 minutes after starting the protocol run Sample reagent Pl2. 0 uL 6 Elution Elution Buffer 900 uL 4 Place a Thermo Scientific KingFisher Flex 24 tip comb on a Tip plate KingFisher Flex 24 5 deep well plate Start the Blood_gDNA_2mL_Flex protocol with the KingFisher Flex 24 and load the plates according to the instructions on the KingFisher display After all the plates have been loaded into the instrument the protocol will begin 6 Add the MagJET Magnetic Beads resuspended well by vortexing and Binding Buffer supplemented with ethanol see page 5 to the Sample plates when the KingFisher Flex24 pauses at the dispense step after the lysis step approximately 5 minutes after Starting the protocol run Pals Plate type Plate name Content Sampleweagent number volume per well Magnetic beads 70 uL 1 Sample 1 KingFisher Flex 24 Binding Buffer 1600uL deep well plate Magnetic beads 70 uL 2 Sample 2 i Binding Buffer 1600 uL 7 Place the Sample plates back into the instrument and press Start After the pause the protocol will continue to the end 8 After the run is finished remove the plates and transfer the eluate which contains the purified DNA to a clean and new tube then close immediately When the protocol is completed remove the plates according to the instructions on the KingFisher display and turn off the instrument Transfer the eluate which contains the purified DNA to a new clean tube and then close immediately Use the purified DNA immediat
3. 6 plate Wash 2 Wash Buffer 2 1000 uL 5 KingFisher Flex 96 KF plate Elution Elution Buffer 150 uL 6 KingFisher Flex 96 KF plate Tip plate 4 Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on an empty Tip plate KingFisher Flex 96 KF plate 5 Start the Blood_gDNA_200uL_Flex protocol with the KingFisher Flex 96 and load the plates according to the instructions on the KingFisher display After all the plates have been loaded into the instrument the protocol will begin 7 6 Add the MagJET Magnetic Bead suspension resuspended well by vortexing and Binding Buffer supplemented with ethanol see page 5 to the Sample plate when the KingFisher Flex pauses at the dispense step after the lysis step approximately 5 minutes after starting the protocol run Plate Sample reagent mber Plate type Plate name Content volume per well PEY Magnetic beads 25 uL 1 Microtiter deep well 96 plate Sample Binding Buffer 400 uL 7 Place the Sample plate back into the instrument and press Start After the pause the protocol will continue to the end 8 When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and turn off the instrument Use the purified DNA immediately in downstream applications or store at 20 C Protocol B Instructions for genomic DNA purification from 1 mL of whole blood using KingFisher Flex and 24 deep w
4. A_2mL_Flex protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 1 User Manual The protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web page on www thermoscientific com onebio Obtain six empty Thermo Scientific KingFisher Flex 24 deep well plates and a Tip plate Prepare the Sample plate 1 and 2 KingFisher Flex 24 deep well plates The 2 mL blood sample is divided into two different plates Sample plate 1 and 2 Pipette 1 mL sample into the well on Sample plate 1 and 1 mL sample into the corresponding well on Sample plate 2 For example a 2 mL blood sample is divided into wells A1 on Sample plate 1 and A1 on Sample plate 2 Add the following reagents to the Sample plates 1 and 2 and leave the plates at room temperature while the other plates are being filled knh Plate type Plate name Content aa Jeti Blood sample 1000 uL 1 Sample 1 Lysis Buffer 500 uL KingFisher Flex 24 deep Proteinase K 60 uL well plate Blood sample 1000 uL 2 Sample 2 Lysis Buffer 500 uL Proteinase K 60 uL 3 Fill the other plates as follows ee Plate type Plate name Content ar lesa 3 Wash 1_1 Wash Buffer 1 4000 uL 4 KingFisher Flex 24 deep Wash 1_2 Wash Buffer 1 4000 uL 5 well plate Wash 2 Wash Buffer 2 400
5. PRODUCT INFORMATION Thermo Scientific MagJET Whole Blood Genomic DNA Kit K2741 K2742 Read Storage information p 4 upon receipt and store the kit components appropriately www thermoscientific com onebio Lot 00000000 Expiry Date 00 0000 CERTIFICATE OF ANALYSIS Thermo Scientific MagJET Whole Blood Genomic DNA Kit is qualified by isolating genomic DNA from 200 uL of frozen human whole blood following the protocol outlined in the manual The quality of isolated DNA is evaluated spectrophotometrically and by agarose gel electrophoresis The purified DNA has an A260 A2s0 ratio of 1 8 0 2 The functional quality of purified DNA is evaluated by digestion with restriction endonucleases Quality authorized by ZA Jurgita ilinskien Rev 1 o pp O o O Contents page COMPONENTS OF TIE Wess acces vinsabvancatnnesventadnidadvievinitcensatutecawentudbrnea EATER E 4 STORAGE sires cris k toe te atacab dain ad aircon nce nates tat bale uated agai A a 4 DESCRIPTION roninas eaa an eree narena eean Ieo OaD anon Eai 4 PSIG Re E a r a AN 4 IMPORTANT NOTES snui nanara an N a cts eat Na AT gah 5 ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED s sssssesssssesrsssesrssserrssrrrssrsrrsnerrsnrerrenerns 5 PROTOCOL SELEC HON GUIDE cinnin anaia a ia ia eint 6 GENOMIC DNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS c cece 7 Protocol A Instructions for genomic DNA purification from 200 uL of whole
6. ate name and type Row Row name Content volume per well Blood DNA plate 1 i ee Magnetic beads 100 uL KingFisher Flex 24 deep well plate amp Binding Buffer 1840 yL 6 Place the Blood DNA plate 1 back into the instrument and press Start After the pause the protocol will continue to the end 7 When the protocol is complete remove the plate and elution strip according to the instructions on the KingFisher Duo display and turn off the instrument Transfer the eluate which contains the purified DNA to a new clean tube and then close immediately The purified DNA is ready for use in downstream applications Protocol F Instructions for genomic DNA purification from 2 mL of whole blood using KingFisher Duo with 6 pin magnet head and 24 deep well plates Note e When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time prepare working solutions of Wash Buffer 1 Wash Buffer 2 and Binding Buffer as described on page 5 e Transfer the Blood_gDNA_2mL_Duo protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 1 User Manual The protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web page on www thermoscientific com onebio e Ensure that you are using the KingFisher Duo 6 pin magnet head and heating block Obtain two empty Thermo Sc
7. blood using KingFisher Flex 96 and Microtiter deep well 96 plates ccsssssssssssessssssssssssssesessscsessesssssessesseseeeseseeesssssesssasens 7 Protocol B Instructions for genomic DNA purification from 1 mL of whole blood using KingFisher Flex and 24 deep well platessa aera tecteeucna tical ch enti tadtns hath ia venentetnnuna tcl thas ta 9 Protocol C Instructions for DNA purification from 2 mL of whole blood using KingFisher Flex and 24 d ep w ll plates zz ronrecn e e uaa a N rt Sau ee 11 Protocol D Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Duo with 12 pin magnet head and Microtiter deep well 96 plate cesses 13 Protocol E Instructions for genomic DNA purification from 1 mL of whole blood using KingFisher Duo with 6 pin magnet head and 24 deep well plates 00 ccccccesecseseceseeesseetesseetssneeessneeeseneeesenees 15 Protocol F Instructions for genomic DNA purification from 2 mL of whole blood using KingFisher Duo with 6 pin magnet head and 24 deep well plates 00 cccccessecesseceseeesestsseetssneseseneenseneeeeenees 17 Protocol G Instructions for manual genomic DNA purification from 200 uL of whole blood 19 Protocol H Instructions for DNA purification from dried blood ceseecsessecsesestssestssestssneetseneeesenees 20 Protocol Instructions for DNA purification from buffy coat oo ceseeccescessessesteseseseetssneetssneeeseneeesenees 20 Protocol J Instruc
8. cid purification workflow The following selection guide summarizes available protocols depending on starting sample volume throughput and sample processing type Automation protocols are optimized for KingFisher Flex and KingFisher Duo instruments Protocol selection guide o s 5 Leloe _ 2 Sample 5 se s2 S MagJET Sample type S 5 23 5 3 S Q purification Page volume of igluo gg 2 2 2 2 2 protoco lt lt 96 e Protocol A page 7 ark 12 Protocol D 13 Fresh or frozen 2 l OISE page whole blood ioi 24 e l Protocol B page 9 m treated with 6 e Protocol E page 15 EDTA or 24 e Protocol C page 11 citrate 2mL 6 e Protocol F page 17 200 uL variable e Protocol G page 19 Dried blood variable e e e Protocol H page 20 1 5 mL of Buffy coat whole variable e e e Protocol page 20 blood Bone marrow 20 uL variable e o e Protocol J page 20 Urine 0 5 mL variable e e e Protocol K page 21 Swabs variable e o e Protocol L page 21 GENOMIC DNA PURIFICATION PROTOCOLS AND PIPETTING INSTRUCTIONS Protocols A G pages 7 19 are recommended for genomic DNA purification from fresh or frozen whole blood treated with EDTA or citrate Note Samples preserved with heparin are not compatible with the kit For genomic DNA purification from dried blood buffy coat bone marrow urine or swabs follow Protocols H L pages 20 21 Protocol A Instructions f
9. dministration to humans or animals Please refer to www thermoscientific com onebio for Material Safety Data Sheet of the product 2013 Thermo Fisher Scientific Inc All rights reserved All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries 23
10. ell plates Note 1 2 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time prepare working solutions of Wash Buffer 1 Wash Buffer 2 and Binding Buffer as described on page 5 Transfer the Blood_gDNA_1mL_Flex protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 1 User Manual The protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web page on www thermoscientific com onebio Obtain five empty Thermo Scientific KingFisher Flex 24 deep well plates and Tip plate Prepare the Sample plate KingFisher Flex 24 deep well plate Add the following reagents to the Sample plate and leave the plate at room temperature while the other plates are being filled eee Plate type Plate name Content oa eet oe Blood sample 1000 uL 1 E ie geep Sample Lysis Buffer 500 uL Proteinase K 60 uL 3 Fill the other plates as follows Enea Plate type Plate name Content it ato 2 Wash 1_1 Wash Buffer 1 3700 uL 3 KingFisher Flex 24 deep Wash 1_2 Wash Buffer 1 3700 uL 4 well plate Wash 2 Wash Buffer 2 3700 uL 5 Elution Elution Buffer 900 uL 4 Place a Thermo Scientific KingFisher Flex 24 tip comb on a Tip plate empty KingFisher Flex 24 deep well plate Start the Blood_gDNA_1mL_Flex protoco
11. ely in downstream applications or store at 20 C Protocol D Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Duo with 12 pin magnet head and Microtiter deep well 96 plate Note 2 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time prepare working solutions of Wash Buffer 1 Wash Buffer 2 and Binding Buffer as described on page 5 Transfer the Blood_gDNA_200uL_Duo protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 1 User Manual The protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web page on www thermoscientific com onebio Ensure you are using the KingFisher Duo 12 pin magnet head and heating block Obtain one empty Thermo Scientific Microtiter 96 deep well plate and one Thermo Scientific KingFisher Duo elution strip Prepare the Blood DNA plate Microtiter deep well 96 plate Add the following reagents to the rows Note that row B is reserved for the tip and should be left empty Note that rows C D and H are left empty Plate name and type Row Row name Content m lap a Blood sample 200 uL A Sample Lysis Buffer 100 uL Proteinase K 20 uL B Tip 12 tip comb Empty Blood DNA plate C Empty Empty Empty Microtiter deep we
12. ent and press Start After the pause the protocol will continue to the end 8 When the protocol is complete remove the plate and elution strip according to the instructions on the KingFisher Duo display and turn off the instrument Transfer the eluate which contains the purified DNA to a new clean tube and then close immediately The purified DNA is ready for use in downstream applications Protocol E Instructions for genomic DNA purification from 1 mL of whole blood using KingFisher Duo with 6 pin magnet head and 24 deep well plates Note e When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time prepare working solutions of Wash Buffer 1 Wash Buffer 2 and Binding Buffer as described on page 5 e Transfer the Blood_gDNA_1mL_Duo protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 1 User Manual The protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web page on www thermoscientific com onebio e Ensure that you are using the KingFisher Duo 6 pin magnet head and heating block Obtain two empty Thermo Scientific KingFisher Flex 24 deep well plates 2 Prepare the Blood DNA plates 1 and 2 Add the following reagents to the Blood DNA plates 1 and 2 KingFisher Flex 24 deep well plates Pipette 1 mL sample into the
13. g equipment IMPORTANT NOTES e Add the indicated volumes of ethanol 96 100 to Binding Buffer concentrated Wash Buffer 1 and 2 prior to first use 96 preps 384 preps Binding Wash Wash Binding Wash Wash Buffer Buffer 1 Buffer 2 Buffer Buffer 1 Buffer 2 Concentrated p aia 100mL 40mL 90mL 100mL 40mL buffer Ethanol 96 100 23 mL 100 mL 200 mL 90 mL 100 mL 200 mL Total volume 46 mL 200 mL 240 mL 180 mL 200 mL 240 mL After preparing each solution mark the bottle to indicate that this step has been completed e Check all solutions in the kit for any salt precipitation before each use Re dissolve any precipitates by warming the solution at 37 C and then equilibrate to room temperature 15 25 C e Wear gloves when handling the Lysis Buffer Binding Buffer and Wash Buffer 1 as these reagents contain irritants see page 22 for SAFETY INFORMATION ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED Pipettes and pipette tips 1 5 mL tubes Disposable gloves 96 100 ethanol molecular biology grade Automatic magnetic particle processor and consumables or Magnetic particle processing rack PROTOCOL SELECTION GUIDE The MagJET Whole Blood Genomic DNA Kit provides optimized protocols for genomic DNA purification from different amounts of starting material 200 uL 1 mL and 2 mL The kit is compatible with automated and manual sample processing allowing low to high throughput nucleic a
14. h ethanol see page 5 Resuspend the magnetic beads by vortexing briefly spin to remove drops from inside of the lid Place the tube on the magnetic rack and let the magnetic beads collect at the magnet for 2 3 minutes Discard the supernatant by using a pipette Carefully eliminate supernatant droplets using a small pipette tip and make sure no droplets are left on the tube wall Note Make sure that all the supernatant is completely removed in the last washing step If there are any visible droplets incubate at room temperature 15 25 C for 1 minute 6 Remove the magnetic rack and add 150 uL Elution Buffer Resuspend the magnetic beads by vortexing incubate for 5 minutes at 72 C place the tube on the magnetic rack and let the magnetic beads collect at the magnet for 2 3 minutes While on the magnetic rack transfer the eluate which contains the purified DNA to a new clean tube then close immediately Use the purified DNA immediately in downstream applications or store at 20 C Protocol H Instructions for DNA purification from dried blood 1 Cut out the section of filter containing the dried blood sample and place into a microcentrifuge tube Add 200 uL of 0 9 w v NaCl and incubate 5 10 minutes at room temperature 15 25 C For manual purification proceed to Step 1 of the Protocol G Instructions for manual genomic DNA purification from 200 uL of whole blood on page 19 For automated purificati
15. ientific KingFisher Flex 24 deep well plates 2 Prepare the Blood DNA plates 1 and 2 by adding the following reagents to the Blood DNA plates 1 and 2 KingFisher Flex 24 deep well plates The 2 mL blood sample is divided into two different wells of rows A and B of the Blood DNA plate 1 Pipette 1 mL sample into the well of row A and 1 mL sample into corresponding well of row B A 2 mL blood sample is divided for example into wells A1 and B1 of the Blood DNA plate 1 3 Add the following reagents to the rows Note that row C of the Blood DNA plate 1 is reserved for the tip and should be left empty Plate name and type Row Row name Content 5 Leathe Blood sample 1000 uL A Sample 1 Lysis Buffer 500 uL EE Proteinase K 60 uL NOAE RAP AIE Blood sample 1000 uL ao as B Sample 2 Lysis Buffer 500 uL Proteinase K 60 uL C Tip 12 tip comb Empty D Wash 1_1 Wash Buffer 1 4800 uL A Elution Elution Buffer 900 uL KingFisher Flex 24 deepwel E Wash22__ Wash Buffer2 5000 uL a pwei Wash21 Wash Buffer 2 5000 pL D Wash 1_2 Wash Buffer 1 4800 uL 4 Place a Thermo Scientific KingFisher Duo 6 tip comb into row C of the Blood DNA plate 1 5 Switch on the KingFisher Duo and make sure that you are using the KingFisher Duo 6 pin magnet head and heating block Start the Blood_gDNA_2mL_Duo protocol Insert the Blood DNA plates into the instrument as indicated on the KingFisher Duo display Afte
16. l with the KingFisher Flex 24 and load the plates according to the instructions on the KingFisher display After all the plates have been loaded into the instrument the protocol will begin Add the MagJET Magnetic Beads resuspended well by vortexing and Binding Buffer supplemented with ethanol see page 5 to the Sample plate when the KingFisher Flex pauses at the dispense step after the lysis step approximately 10 minutes after starting the protocol run Plate number 1 Plate type KingFisher Flex 24 deep well plate Plate name Sample Sample reagent Content volume per well Magnetic beads 70 uL Binding Buffer 1600 uL 7 Place the Sample plates back into the instrument and press Start After the pause the protocol will continue to the end 8 When the protocol is completed remove the plates according to the instructions on the KingFisher display and turn off the instrument Transfer the eluate which contains the purified DNA to a new clean tube and then close immediately Use the purified DNA immediately in downstream applications or store at 20 C Protocol C Instructions for DNA purification from 2 mL of whole blood using KingFisher Flex and 24 deep well plates Note N When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time prepare working solutions of Wash Buffer 1 Wash Buffer 2 and Binding Buffer as described on page 5 Transfer the Blood_gDN
17. ll 96 plate D Empty Empty Empty E Wash 1_1 Wash Buffer 1 800 uL F Wash 1_2 Wash Buffer 1 800 uL G Wash 2 Wash Buffer 2 1000 uL H Empty Empty Empty 3 Fill the KingFisher Duo elution strip as follows Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user and the Elution Buffer is pipetted into the correct wells Elution strip Content Reagent volume per well KingFisher Duo elution strip Elution Buffer 130 uL 4 Place a Thermo Scientific KingFisher Duo 12 tip comb into row B on the Blood DNA plate 5 Switch on the KingFisher Duo instrument and ensure you are using the KingFisher Duo 12 pin magnet head and heating block Start the Blood_gDNA_200uL_Duo protocol Insert the Blood DNA plate and elution strip into the instrument as indicated on the KingFisher Duo display Make sure that the elution strip is placed in the correct direction into the elution block Ensure that the perforated end is facing towards the user 13 6 Add the MagJET Magnetic beads resuspended well by vortexing and Binding Buffer supplemented with ethanol see page 5 to row A of the Blood DNA plate Sample reagent Plate name and type Row Row name Content volume per well Blood DNA plate Magnetic beads 25 UL eke A Sample a Microtiter deep well 96 plate Binding Buffer 400 uL 7 Place the Blood DNA plate back into the instrum
18. ment Protocols for the other automated pipetting platforms should be optimized for each platform and sample used To enable protocol optimization all buffers are available to purchase separately Note When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time prepare working solutions of Wash Buffer 1 Wash Buffer 2 and Binding Buffer as described on page 5 1 Add 20 uL Proteinase K and 100 uL Lysis Buffer to a 200 uL blood sample Vortex 30 s at maximum speed and incubate 3 minutes at room temperature 15 25 C Vortex 15 s to mix and briefly spin to remove drops from inside of the lid Add 25 uL MagJET Magnetic beads resuspended well by vortexing and 400 uL Binding Buffer Supplemented with ethanol see page 5 Resuspend the magnetic beads by vortexing briefly spin to remove droplets from inside of the lid Place the tube on the magnetic rack and let the magnetic beads collect at the magnet for 3 minutes Discard the supernatant by using a pipette Remove the magnetic rack and add 800 uL Wash Buffer 1 supplemented with ethanol see page 5 Resuspend the magnetic beads by vortexing briefly spin to remove drops from inside of the lid Place the tube on the magnetic rack and let the magnetic beads collect at the magnet for 2 3 minutes Discard the supernatant by using a pipette 4 Repeat step 3 using 800 uL of Wash Buffer 1 5 Remove the magnetic rack and add 1000 uL Wash Buffer 2 supplemented wit
19. nts of labelling sodium perchlorate Risk phrases 22 Harmful if swallowed 9 Explosive when mixed with combustible material Safety phrases 17 Keep away from combustible material 23 Do not breathe gas fumes vapour spray 27 Take off immediately all contaminated clothing 36 Wear suitable protective clothing 60 This material and its container must be disposed of as hazardous waste Wash Buffer 1 conc for MagJET Blood gDNA Kit O Oxidizing Hazard determining components of labelling sodium perchlorate Risk phrases 9 Explosive when mixed with combustible material Safety phrases 17 Keep away from combustible material 27 Take off immediately all contaminated clothing 60 This material and its container must be disposed of as hazardous waste 22 x Proteinase K Xn Harmful Hazard determining components of labelling Proteinase Tritirachium album serine Risk phrases 42 May cause sensitisation by inhalation Safety phrases 23 Do not breathe gas fumes vapour spray 36 Wear suitable protective clothing 45 In case of accident or if you feel unwell seek medical advice immediately show the label where possible 60 This material and its container must be disposed of as hazardous waste PRODUCT USE LIMITATION This product is developed designed and sold exclusively for research purposes and in vitro use only The product was not tested for use in diagnostics or for drug development nor is it suitable for a
20. on using KingFisher Flex 96 or KingFisher Duo Instruments 3 proceed to Step 1 of corresponding Protocol A Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Flex 96 and Microtiter deep well 96 plates on page 7 or Protocol D Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Duo with 12 pin magnet head and 96 deep well plate on page 13 Protocol I Instructions for DNA purification from buffy coat Centrifuge 1 5 mL of whole blood at 2 500 x g 5 000 rpm for 10 minutes at room temperature Three layers should be visible 2 Remove upper clear layer by aspiration 3 Collect approximately 200 uL of intermediate layer using an automatic pipette For manual purification proceed to Step 1 of the Protocol G Instructions for manual genomic DNA purification from 200 uL of whole blood on page 19 For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments 4 proceed to Step 1 of corresponding Protocol A Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Flex 96 and Microtiter deep well 96 plates on page 7 or Protocol D Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Duo with 12 pin magnet head and 96 deep well plate on page 13 Protocol J Instructions for DNA purification from bone marrow 1 Harvest 20 uL of fresh or frozen b
21. one marrow For manual purification proceed to Step 1 of the Protocol G Instructions for manual genomic DNA purification from 200 uL of whole blood on page 19 For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments proceed to Step 1 of corresponding Protocol A Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Flex 96 and Microtiter deep well 96 plates on page 7 or Protocol D Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Duo with 12 pin magnet head and 96 deep well plate on page 13 20 Protocol K Instructions for DNA purification from urine 1 Add 0 5 mL of 0 5 M EDTA to 4 5 mL of urine final concentration 50 mM Centrifuge 10 minutes at 800 x g 3 000 rpm Discard the supernatant 2 3 4 Resuspend the pellet in 200 uL of 0 9 w v NaCl For manual purification proceed to Step 1 of the Protocol G Instructions for manual genomic DNA purification from 200 uL of whole blood on page 19 For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments proceed to Step 1 of corresponding Protocol A Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Flex 96 and Microtiter deep well 96 plates on page 7 or Protocol D Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Duo with 12 pin magne
22. or genomic DNA purification from 200 uL of whole blood using KingFisher Flex 96 and Microtiter deep well 96 plates Note 2 When using the MagJET Whole Blood Genomic DNA Purification Kit for the first time prepare working solutions of Wash Buffer 1 Wash Buffer 2 and Binding Buffer as described on page 5 Transfer the Blood_gDNA_200uL_Flex protocol file to the KingFisher instrument before first use The instructions for transferring the protocol can be found in Chapter 4 Using the software in the Bindlt Software for KingFisher Instruments version 3 1 User Manual The protocol files for MagJET Whole Blood Genomic DNA Kit can be found on product web page on www thermoscientific com onebio Obtain four empty Thermo Scientific Microtiter deep well 96 plates and two empty Thermo Scientific KingFisher Flex 96 KF plates Prepare the Sample plate Microtiter deep well 96 plate Add the following reagents to the Sample plate and leave the plate at room temperature while the other plates are being filled ile Plate type Plate name Content a ee Blood sample 200 uL 1 Microtiter deep well 96 plate Sample Lysis Buffer 100 uL Proteinase K 20 uL 3 Prepare the other plates as follows Dp Plate type Plate name Content it loeti ah 2 Microtiter deep well 96 plate Wash 1_1 Wash Buffer 1 800 uL 3 Microtiter deep well 96 plate Wash 1_2 Wash Buffer 1 800 uL 4 Microtiter deep well 9
23. performance High binding capacity uniform particle size and rapid magnetic response of MagJET magnetic beads makes the technology ideal for high throughput automatic nucleic acid purification as well as for manual nucleic acid purification by low sample throughput users The resulting high quality DNA is free of proteins nucleases and other contaminants or inhibitors can be used in a wide range of downstream applications such as PCR qPCR and other enzymatic reactions PRINCIPLE The MagJET Whole Blood Genomic DNA Kit uses a highly efficient MagJET magnetic particle based technology for nucleic acid purification The whole nucleic acid isolation process combines simple steps of sample lysis DNA binding to the magnetic beads washing and elution Purification protocols optimized for automated KingFisher instruments utilize a high throughput magnetic bead transfer technique where magnetic beads are transferred through different reagent plates containing lysis binding wash and elution reagents This enables high throughput nucleic acid purification and eliminates multiple pipetting steps Alternatively protocol is available where instead of magnetic particles buffers and other reagents are transferred in each of the protocol steps while magnetic beads remain captured on the wall of the tube with the help of a magnetic rack This allows the kit to be used in various throughput applications using a magnetic rack and manual or automated pipettin
24. r both plates have been loaded into the instrument the protocol will begin 6 Add the MagJET Magnetic beads resuspended well by vortexing and Binding Buffer supplemented with ethanol see page 5 to rows A and B of the Blood DNA plate 1 when the KingFisher Flex pauses at the dispense step after the lysis step approximately 10 minutes after starting the protocol run Sample reagent Plate name and type Row Row name Content volume per well Magnetic beads 70 uL Blood DNA plate 1 oe welt Binding Buffer 1600 uL KingFisher Flex 24 deep well plate B Sample 1 Magnetic beads 70 uL Binding Buffer 1600 uL 7 Place the Blood DNA plate 1 back into the instrument and press OK After the pause the protocol will continue to the end 8 When the protocol is complete remove the plates according to the instructions on the KingFisher Duo display and turn off the instrument Transfer the eluate which contains the purified DNA to a new clean tube and then close immediately The purified DNA is ready for use in downstream applications Protocol G Instructions for manual genomic DNA purification from 200 uL of whole blood The following protocol is based on transfer of liquids by pipetting through different purification steps rather than magnetic bead transfer as in KingFisher automatic protocols This allows the kit to be used in various throughput applications using magnetic rack and manual or automated pipetting equip
25. t head and 96 deep well plate page 13 Protocol L Instructions for DNA purification from swabs 1 To collect a sample scrape the swab 5 6 times against the inside cheek 2 Swirl the swab for 30 60 s in 200 uL of 0 9 w v NaCl For manual purification proceed to Step 1 of the Protocol G Instructions for manual genomic DNA purification from 200 uL of whole blood on page 19 For automated purification using KingFisher Flex 96 or KingFisher Duo Instruments 3 proceed to Step 1 of corresponding Protocol A Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Flex 96 and Microtiter deep well 96 plates on page 7 or Protocol D Instructions for genomic DNA purification from 200 uL of whole blood using KingFisher Duo with 12 pin magnet head and 96 deep well plate page 13 21 SAFETY INFORMATION Xx Lysis Buffer for MagJET Blood gDNA Kit Xn Harmful Hazard determining components of labelling guanidinium chloride Risk phrases 22 Harmful if swallowed 36 38 Irritating to eyes and skin Safety phrases 23 Do not breathe gas fumes vapour spray 26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice 36 37 Wear suitable protective clothing and gloves 60 This material and its container must be disposed of as hazardous waste Binding Buffer for MagJET Blood gDNA Kit Xn Harmful O Oxidizing Hazard determining compone
26. tions for DNA purification from bone MarrOW c ccecseseceesecteseetssestssneetseneeeeeees 20 Protocol K Instructions for DNA purification from urine cceccccesecsessessesseesesseesesneetssneeessneeesenseesenees 21 Protocol L Instructions for DNA purification from swabs cccccscesseseseseeesseeesneetssneenseneeessneeesenees 21 SAFETY INFORMATION sisemine en E A E 22 COMPONENTS OF THE KIT MagJET Whole Blood Genomic DNA Kit geet lee 96 preps 384 preps Proteinase K 2x1 0mL 8x 1 0 mL Lysis Buffer for MagJET Blood gDNA Kit 15 mL 60 mL MagJET Magnetic Beads 2x1 4mL 10 6 mL Binding Buffer conc for MagJET Blood gDNA Kit 23 mL 90 mL Wash Buffer 1 conc for MagJET Blood gDNA Kit 100 mL 4 x 100 mL Wash Buffer 2 conc for MagJET Blood gDNA Kit 40 mL 2 x 40mL Elution Buffer 30 mL 3 x 30mL STORAGE Proteinase K solution is stable at room temperature as long as the vial remains sealed After opening store at 20 C MagJET Magnetic Beads should be stored at 4 C Other components of the kit should be stored at room temperature 15 25 C DESCRIPTION The MagJET Whole Blood Genomic DNA Kit is designed for fast and efficient purification of genomic DNA from fresh or frozen whole blood treated with EDTA or citrate as well as blood fractions Note Samples preserved with heparin are not compatible with this kit The kit utilizes paramagnetic bead technology enabling high yields and robust
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