Product information: MagJET Genomic DNA Kit, #K2721, #K2722
Contents
1.2. Grind up to 20 mg of mammalian tissue 0 5 cm mouse tail clip or up to 20 mg of insect in liquid nitrogen using a mortar and pestle Alternatively cut the tissue into small pieces or disrupt it using a homogenizer Note use up to 5 mg of spleen and lung tissue High quantities of DNA 2 4 ug mg tissue from indicated tissues result in high viscosity of eluates and contamination by magnetic beads Using 200 uL instead of 150 uL Elution Buffer is also recommended in step 5 when isolating DNA from 5 mg spleen or lung tissues N Collect the material into a 2 0 mL microcentrifuge tube not provided prefilled by 200 uL of Digestion Solution and mix thoroughly by vortexing for 5 10 s Add 20 uL of Proteinase K Solution and mix thoroughly by vortexing or pipetting to obtain a uniform suspension Se Incubate the sample at 56 C During incubation vortex the vial occasionally or use a shaking water bath rocking platform or thermomixer Suggested optimal incubation times for different types of tissue are provided in the table below Type of tissue Suggested incubation times Liver 1 hour Heart 1 hour Rodent tail 1 hour Muscle 1 hour Brain 1 hour Spleen 2 hours Lung 2 hours Kidney 2 hours Ear 2 hours Skin 3 hours Hair follicles 1 hour 4 Proceed to Step 3 of Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtit
3. PRODUCT INFORMATION Thermo Scientific MagJET Genomic DNA Kit K2721 K2722 Read Storage information p 4 upon receipt and store kit components appropriately www thermoscientific com onebio K2721 K2722 Lot 00000000 Expiry Date 00 0000 CERTIFICATE OF ANALYSIS Thermo Scientific MagJET Genomic DNA Kit is qualified by isolating genomic DNA from 5 mg of mouse liver following the protocols outlined in the manual The quality of purified genomic DNA is evaluated spectrophotometrically and by agarose gel electrophoresis The purified genomic 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 Rev 1 Ill 45 Jurgita Zilinskien CONTENTS page COMPONENTS OF THE KIT E 4 STORAGE stvccssaasleathillescesscccaleantcesuonnseteanitinss eolian vaaata Mi oottaa a Eai aeeiiaii iiaeo iea as 4 DESCRIPTION inahin enana N nederdel a a aaan 4 PRINCIPLE sisisi iana a iiie e E A i diiio 4 IMPORTANT NOTES camerier e e EEA EER 5 ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED 0 sccseesseesstesseessessneestesseesseesneesneesnesneesneesnseeneenes 6 STARTING MATERIAL HANDLING AND STORAGE l PROTOCOL SELECTION GUIDE 2 cscisteccunuiianncnudiendtie dian nun i i E E GENOMIC DNA PURIFICATION PROTOCOLS AND PIPETTING INSRUCTIONS cescsssesseestesesseeeseesneees 8 Protocol A Instructions for genomic DNA puri
4. 108 cells 7 11 ug 14 HeLa cells 108 cells 7 11 ug 15 Jurkat cells 108 cells 6 12 ug 16 COS 7 cells 10 cells 3 5 ug 17 Human blood leucocytes leucocytes collected from 1 mL of blood 13 17g IMPORTANT NOTES e Add the indicated volume of ethanol 96 100 to Wash Buffer 1 conc and Wash Buffer 2 conc prior to first use 96 preps 384 preps Wash Buffer1 Wash Buffer2 Wash Buffer1 Wash Buffer 2 Concentrated buffer 25 mL 50 mL 50 mL 50 mL Ethanol 96 100 75 mL 150 mL 150 mL 150 mL Total volume 100 mL 200 mL 200 mL 200 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 and Wash Buffer 1 as these reagents contain irritants see page 25 for SAFETY INFORMATION 5 ADDITIONAL MATERIALS AND EQUIPMENT REQUIRED Pipettes and pipette tips 1 5 2 mL plastic tubes Vortex Microcentrifuge Disposable gloves 96 100 ethanol molecular biology grade Equipment for sample disruption and homogenization depending on the method chosen e Mortar and pestle e Homogenizer e Automatic magnetic particle processor and consumables e Magnetic particle processing rack Buffers For mammalian cell lysate p
5. 2 Collect cells by centrifugation at 4 000 x g for 30 minutes at room temperature 3 Centrifuge at 1 700 x g for 5 minutes at 4 C 3 Discard the supernatant 4 Carefully remove and discard the supernatant completely without disturbing the visible white leukocytes pellet For manual gDNA purification proceed to Step 2 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 5 proceed to Step 2 of Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 11 For manual gDNA purification proceed to Step 2 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and isects on page 14 For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 4 proceed to Step 2 of Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo a
6. Starting material was not completely digested Tissue samples should be homogenized very thoroughly in liquid nitrogen Samples of cells should be suspended very well in small quantity of TE or Low yield of 0 15 M NaCl solution 30 40 uL before adding Digestion Solution purified DNA lsopropanol was not added to the lysate Make sure that the isopropanol was added to the lysate before transferring the sample to the plate of magnetic particle processor Magnetic beads have to be resuspended well by vortexing before adding to isopropanol Ethanol was not added to Wash Buffer s Make sure that ethanol was added to Wash Buffer 1 and Wash Buffer 2 before use Follow the instructions for Wash Buffer preparation on p 5 Sample was frozen and thawed repeatedly Avoid repeated freeze thaw cycles of the samples Use a new sample for Purifi DNA isolation Perform extractions from fresh material when possible urified DNA ue is degraded Inappropriate sample storage conditions l l Store mammalian tissues at 70 C and bacteria at 20 C or 70 C until use For long term storage clinical samples urine saliva should be stored at 20 C RNA RNase A treatment was not carried out contamination Carry out RNase A treatment step described in the purification procedure 23 Low A260 A280 Some magnetic particles are left in the elution centrifuge eluates at full ratio from UV speed for 1 minute and transfer supernatant to a new
7. beads for 2 minutes and discard remaining supernatant Remove the magnetic rack and add 150 uL Elution Buffer Resuspend the magnetic beads by vortexing incubate tubes in thermomixer at 72 C 600 700 rpm for 5 9 minutes Spin down the tube to collect all the drops from the walls of the tube 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 10 new clean tube then close immediately For long term storage of DNA eluting in Elution Buffer and storing at 20 C is recommended 14 Protocol F Instructions for manual genomic DNA purification from up to 10 cultured mammalian cells Note When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 Step Procedure Collection of cultured mammalian cells a Suspension cells collect up to 106 cells in a centrifuge tube Pellet cells by centrifugation for 5 minutes at 250 x g Discard the supernatant 1 b Adherent cells remove growth medium from a culture plate containing up to 108 cells Detach the cells from the culture plate by trypsinization in an appropriate volume of PBS Transfer the cells to a microcentrifuge tube and pellet by centrifugation for 5 minutes at 250 x g Discard the supernatant Resuspend the cells collected in st
8. platform or thermomixer for 20 30 minutes until the cells are completely lysed Obtain one empty Thermo Scientific Microtiter deep well 96 plate and one Thermo Scientific KingFisher Duo elution strip Add 20 uL of RNase A Solution mix by vortexing then incubate for 10 minutes at room temperature add 300 uL of Lysis Buffer vortex 5 10 seconds and transfer lysates to Sample row prefilled by 400 uL of isopropanol and 25 uL of magnetic beads Prepare the DNA plate Microtiter deep well 96 plate according to the instructions below Add the following reagents to the rows Note that row H is reserved for the tip and should be left empty Note that rows E F and G are left empty Sample Plate name and type Row Row name Content reagent volume per well Lysed sample 580 uL A Sample Isopropanol 100 400 uL Magnetic Beads 25 uL Wash Buffer 1 B Wash supplemented with ethanol 800 uL DNA plate Microtiter deep well 96 Wash Buffer 2 plate C i Washiz d supplemented with ethanol 800 uL Wash Buffer 2 De hase supplemented with ethanol agree E Empty Empty Empty F Empty Empty Empty G Empty Empty Empty H Tip comb 12 Tip comb Resuspend Magnetic Beads well by vortexing before use 6 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 toward
9. using a mortar and pestle Alternatively cut the tissue into small pieces or disrupt it using a homogenizer Note For spleen and lung tissue use up to 5 mg High quantities of DNA 2 4 ug mg tissue from indicated tissues result in high viscosity of eluates and contamination by magnetic beads Using 200 uL instead of 150 uL Elution Buffer in step 5 is also recommended when isolating DNA from 5 mg spleen or lung tissues Collect the material into a 2 0 mL microcentrifuge tube not provided prefilled by 200 uL of Digestion Solution and mix thoroughly by vortexing for 5 10 s Add 20 uL of Proteinase K Solution and mix thoroughly by vortexing or pipetting to obtain a uniform suspension Incubate the sample at 56 C During incubation vortex the vial occasionally or use a shaking water bath rocking platform or thermomixer Suggested optimal incubation times for different types of tissue are provided in the table below Type of tissue Suggested incubation times Liver 1 hour Heart 1 hour Rodent tail 1 hour Muscle 1 hour Brain 1 hour Spleen 2 hours Lung 2 hours Kidney 2 hours Ear 2 hours Skin 3 hours Hair follicles 1 hour 4 Proceed to Step 3 of Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 for the further purification Protocol C Instructions for genomic DNA purific
10. After being opened it should be stored at 20 C When the kit is delivered remove the RNase A from the package and store at 20 C MagJET Magnetic Beads should be stored at 4 C For longer use it is recommended to aliquot Elution Buffer as 1 2 mL samples and store at 20 C Other components of the kit should be stored at room temperature 15 25 C DESCRIPTION The MagJET Genomic DNA Kit is designed for fast and efficient purification of genomic DNA from tissue and cell cultures bacteria and yeast as well as from human body samples such as buccal and urogenital swabs urine saliva and hair follicles The kit utilizes paramagnetic bead technology enabling high yields and robust 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 purification for low sample throughput The resulting high quality DNA is free of proteins nucleases and other contaminants or inhibitors and can be used in a wide range of downstream applications such as PCR qPCR or other enzymatic reactions See Table 1 for typical genomic DNA yields from various sources PRINCIPLE The MagJET Genomic DNA Kit uses the 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 b
11. ation from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates Note N Se a on When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 Transfer the Tissue_gDNA_Duo protocol file to the KingFisher Duo as described on page 7 Ensure you are using the KingFisher Duo 12 pin magnet head and heating block Collect the Cultured Mammalian Cells as follows a Suspension cells Pellet up to 10 cells in an appropriate centrifuge tube for 5 min at 300 x g Discard the supernatant Rinse the cells once with PBS to remove residual growth medium Repeat the centrifugation step and discard the supernatant b Adherent cells Remove growth medium from the cells use up 10 cells Rinse the cells once with PBS to remove residual medium Remove and discard PBS Detach the cells from the culture plate by scraping in an appropriate volume of PBS or by trypsinization Transfer the cells into a microcentrifuge tube not included and pellet by centrifugation for 5 min at 300 x g Discard the supernatant Resuspend the collected cells in 40 uL of 0 15 M NaCl solution add 200 uL of Digestion Solution and 20 uL of Proteinase K Solution Mix the cell lysate thoroughly by vortexing or pipetting to obtain a uniform suspension Incubate the sample at 56 C while vortexing occasionally or use a shaking water bath rocking
12. drolysis procedure with Proteinase K up to 6 or 16 hours overnight does not increase yield and quality of purified DNA PROTOCOL SELECTION GUIDE The MagJET Genomic DNA Kit provides optimized protocols for genomic DNA purification from different amounts of starting material up to 10 cells and up to 20 mg tissue The kit is compatible with automated and manual sample processing allowing low to high throughput nucleic acid purification workflow The following selection guide summarizes available protocols depending on starting sample quantity throughput and sample processing type Automation protocols are optimized for KingFisher Flex and KingFisher Duo instruments Transfer the Tissue_gDNA_Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo 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 2 User Manual The protocol files for MagJET Genomic DNA Kit can be found on product web page on www thermoscientific com onebio GENOMIC DNA PURIFICATION PROTOCOLS AND PIPETTING INSRUCTIONS Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates Note e When using the MagJET Genomic DNA Kit for the first time prepare wor
13. eads 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 washing and elution reagents This enables high throughput nucleic acid purification and eliminates multiple pipetting steps Alternatively a protocol is available where buffers and other reagents are transferred in each of the protocol steps while magnetic beads remain captured on the wall of the tube using a magnetic rack This allows the kit to be used for various throughput applications using a magnetic rack and manual or automated pipetting equipment Table 1 Typical genomic DNA yields from various sources Source Quantity DNA yield 5 mg 4 5 ug 1 Mouse heart 20 mg 18 19 ug 5 mg 6 pg 2 Mouse tail homogenized 20 mg 15 ug 5mg 1 ug 3 Mouse tail cut 20 mg 4 5 ug 5 mg 14 15 ug 4 Mouse liver 20 mg 50 ug 5 mg 20 ug 5 Mouse spleen 20 mg 72 75 ug 5mg 9 10 ug 6 Mouse kidney 20 mg 35 38 ug 5 mg 2 5 ug 7 Mouse muscle 20 mg 8 9 ug 5mg 3 ug 8 Mouse brain 20 mg 105 11 ug 5mg 5 Ug 9 Mouse skin 20 mg 18 19 ug 5 mg 9 10 ug 10 Mouse lung 20 mg 36 38 ug 5 mg 9 5 10 ug 11 Mouse ear 20 mg 35 37 ug 12 E coli cells 2 x 10 cells 13 17 ug 13 S cerevisiae
14. emain During incubation vortex the vial occasionally or use a shaking water bath rocking platform or thermomixer Suggested incubation times for different tissue samples are indicated on page 13 Add 20 uL of RNase A Solution mix by vortexing then incubate for 10 min at room temperature Add 300 uL of Lysis Buffer vortex 5 10 seconds and transfer lysate to a tube prefilled with 400 uL of isopropanol and 25 uL of magnetic beads suspension resuspended well by vortexing Mix the tube by vortexing Place the tube on the magnetic rack and let the magnetic beads collect at the magnet for 3 minutes Remove the supernatant using a pipette Remove the tube from the magnetic rack and add 800 uL Wash Buffer 1 supplemented with ethanol see p 5 Resuspend the magnetic beads by vortexing 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 Remove the magnetic rack and add 800 uL Wash Buffer 2 supplemented with ethanol see p 5 Resuspend the magnetic beads by vortexing place the tube on the magnetic rack and let the magnetic beads collect at the magnet for 2 3 minutes Remove the supernatant using a pipette Repeat step 7 using 800 uL of Wash Buffer 2 Make sure that all the supernatant is completely removed in this last washing step If there are still some droplets visible spin down the tube then place it on magnetic rack to collect the
15. ep 1a or 1b in 40 uL of 0 15 M NaCl solution add 200 uL of Digestion Solution and 20 uL of Proteinase K Solution Mix the cell lysate 2 thoroughly by vortexing or pipetting to obtain a uniform suspension Incubate the sample at 56 C while vortexing occasionally or use a shaking water bath rocking platform or thermomixer for 20 30 minutes or until the cells are completely lysed Add 20 uL of RNase A Solution mix by vortexing then incubate for 10 min at room temperature Add 300 uL of Lysis Buffer vortex 5 10 seconds and transfer lysate to a 3 tube prefilled with 400 uL of isopropanol and 25 uL of magnetic beads suspension resuspended well by vortexing Mix the tube by vortexing then incubate for 5 min at room temperature Proceed to Step 5 of Protocol E Instructions for manual genomic DNA purification 4 from up to 20 mg tissue rodent tail and insects on page 14 Protocol G Instructions for genomic DNA purification from gram negative bacterial cultures up to 10 cells Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA_Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Protocol H Instructions for genomic DNA purification from yeast culture up to 108 cells Note e When using the MagJET Genomic DNA Kit for t
16. er deep well 96 plates on page 11 for the further purification Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects This protocol is based on transfer of liquids by pipetting through different purification steps rather than magnetic bead transfer as in KingFisher automatic protocols It allows the kit to be used for various throughput applications using a magnetic rack and manual or automated pipetting equipment Protocols for different automated pipetting equipment should be optimized for each platform as well as the sample type used To enable protocol optimization all buffers are available to purchase separately Note When using the MagJET Genomic DNA kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 Step Procedure Homogenize up to 15 mg of mammalian tissue use up to 5 mg of spleen and lung tissue 0 2 0 4 cm rat or mouse tail clip or up to 20 mg of insect in liquid nitrogen using a mortar and pestle Alternatively cut the tissue into small pieces or disrupt it using a homogenizer Collect the material into a 2 0 mL microcentrifuge tube not provided prefilled by 2 200 uL of Digestion Solution and resuspend Add 20 uL of Proteinase K Solution and mix thoroughly by vortexing or pipetting to obtain a uniform suspension Incubate the sample at 56 C until the tissue is completely lysed and no particles r
17. fication from up to 106 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates cceececccseeseseseesesseseesesesesseeseseesesnsensseseseseeeneeeeed 8 Protocol B Instructions for genomic DNA purification from up to 20 mg of tissue rodent tail and insects using KingFisher Flex 96 and Microtiter deep well 96 plates cccceeceseesesseseesesesessesesessestesteaesteeesteeeeneees 10 Protocol C Instructions for genomic DNA purification from up to 108 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates 0 ceseccseeessesteesesteesseseeseeteseenestesneneetseteaeeneeeeneeneeees 11 Protocol D Instructions for genomic DNA purification from up to 20 mg tissue rodent tail and insects using KingFisher Duo and Microtiter deep well 96 plates eecccccseecesseseesessesteesesessesteseenesteseeeseesteneensseseeneeees 13 Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and INSOCtS ia uisieseceecteshecitectea dean nani aia teiaa aea RA i E iinet 14 Protocol F Instructions for manual genomic DNA purification from up to 108 cultured mammalian cells 15 Protocol G Instructions for genomic DNA purification from gram negative bacterial cultures upto 109 cells iena ateiti eea duane ET RE A aean E E E 15 Protocol H Instructions for genomic DNA purification from yeast culture up to 108 Cells eee 16 Protocol Instruc
18. he first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Before starting e Prepare yeast lysis buffer 1 M sorbitol 0 1 M EDTA pH 7 4 Just prior to use add 0 1 B mercaptoethanol and 5 mg mL Zymolyase 20T Step Procedure Harvest up to 108 yeast cells about 1 mL of overnight culture in a 1 5 or 2 mL 1 microcentrifuge tube by centrifugation for 5 10 seconds at maximum speed 212 000 x g Discard the supernatant 2 Resuspend the pellet in 500 uL of yeast lysis buffer Incubate for 1 hour at 37 C Centrifuge cells for 10 minutes at 3 000 x g Discard the supernatant Resuspend the pellet in 200 uL of Digestion Solution Add 20 uL of Proteinase K 3 Solution and mix thoroughly by vortexing or pipetting to obtain a uniform suspension Incubate the sample at 56 C while vortexing occasionally or use a shaking water bath rocking platform or thermomixer until the cells are completely lysed 45 60 minutes For manual gDNA purification proceed to Step 4 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 4 proceed to Step 3 of Protocol A Instructions for genomic DNA purifica
19. he sample plate as follows after cells have been incubated with Proteinase K add 20 uL of RNase A Solution mix by vortexing then incubate for 10 minutes at room temperature Add 300 uL of Lysis Buffer vortex 5 10 seconds and transfer lysates to Sample plate prefilled by 400 uL of isopropanol and 25 uL of magnetic beads Place a Thermo Scientific KingFisher Flex 96 tip comb for deep well magnets on a Tip Plate empty KingFisher Flex 96 KF plate Start the Tissue_gDNA_Flex protocol on the KingFisher Flex 96 and load the plates according to the KingFisher display After all the plates have been loaded into the instrument the protocol will begin When the protocol is completed remove the plates according to the instructions on the KingFisher Flex display and turn off the instrument The purified gDNA is ready for use in downstream applications Use the purified gDNA immediately or store it at 20 C Protocol B Instructions for genomic DNA purification from up to 20 mg of tissue rodent tail and insects using KingFisher Flex 96 and Microtiter deep well 96 plates Note N w When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 Transfer the Tissue_gDNA_Flex protocol file to the KingFisher Flex as described on page 7 Grind up to 20 mg of mammalian tissue 0 5 cm mouse tail clip or up to 20 mg of insect in liquid nitrogen
20. king solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA_Flex protocol file to the KingFisher Flex as described on page 7 1 Collect the Cultured Mammalian Cells as follows a Suspension cells Pellet up to 10S cells in an appropriate centrifuge tube for 5 min at 300 x g Discard the supernatant Rinse the cells once with PBS to remove residual growth medium Repeat the centrifugation step and discard the supernatant b Adherent cells Remove growth medium from the cells use up 108 cells Rinse the cells once with PBS to remove residual medium Remove and discard PBS Detach the cells from the culture plate by scraping in an appropriate volume of PBS or by trypsinization Transfer the cells into a microcentrifuge tube not included and pellet by centrifugation for 5 min at 300 x g Discard the supernatant Resuspend the cells collected in 40 uL of 0 15 M NaCl solution add 200 uL of Digestion Solution and 20 uL of Proteinase K Solution Mix the cell lysate thoroughly by vortexing or pipetting to obtain a uniform suspension Incubate the sample at 56 C while vortexing occasionally or use a shaking water bath rocking platform or thermomixer until the cells are completely lysed 20 30 minutes 3 Obtain four Thermo Scientific Microtiter deep well 96 plates and one Thermo Scientific KingFisher Flex 96 KF plate 4 Add the following reagents to the plates and leave the plates at room temperature whi
21. lates on page 11 Protocol I Instructions for genomic DNA purification from blood leucocytes Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Before starting e Prepare 1X RBC buffer 10X buffer 1 68 M NH4Cl 2 mM EDTA Step Procedure Protocol J Instructions for genomic DNA purification from urine sediments Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Before starting e Prepare 1X TE buffer 10 mM Tris HCl pH 8 0 1 mM EDTA 1 Add 3 volumes of cold 4 C prepared 1X RBC buffer to fresh blood 0 5 1 mL in plastic tube Step Procedure Mix completely by vortexing and incubate on ice for 4 7 minutes Mix briefly by 2 vortexing two times during incubation The cloudy suspension becomes translucent during incubation indicating lysis of erythrocytes 1 Dilute 20 25 mL morning urine by equal volume of TE buffer or distilled water to prevent precipitation of salts
22. le at 56 C until the tissue is completely lysed and no particles 3 remain During incubation vortex the vial occasionally or use a shaking water bath rocking platform or thermomixer Suggested incubation times for different tissue samples are indicated on page13 21 For manual gDNA purification proceed to Step 4 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 for manual purification For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 4 proceed to Step 3 of Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 11 Note It is recommended to elute DNA in 50 uL of Elution Buffer 22 Protocol O Instructions for genomic DNA purification from milk Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA_Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Step Procedure 1 Add 300 uL of Lysis Buffer to 200 Lof fresh milk
23. le the Sample plate is being prepared N Plate Plate type Plate Content Volume per number name well Isopropanol 100 400 pL 1 Sample Magnetic Beads 25 uL o Wash Buffer 1 supplemented 2 Microtiter deep Wash 1 with ethanol 800 uL well 96 plate Wash 2_1 Wash Buffer 2 supplemented 3 with ethanol SUHI Wash 2_2 Wash Buffer 2 supplemented 4 with ethanol SUHE KingFisher Flex F Elution buffer 5 96 KF plate Elution 150 uL x lt o E rela e _ 2 Sample 25 5 z z MagJET Sample type y P 535l 5 g purification Page 2 Bz E protocol lt lt 96 e Protocol A page 8 i to 10 P eel aie 24 Protocol C page 11 variable e ProtocolF page 15 96 e Protocol B page 10 a sid ae 24 f Protocol D page 13 variable e ProtocolE page 14 j 9 pee ne variable e e e Protocol G page 15 8 Yeast culture nas variable e e Protocol H page 16 Blood collected Bacoees from 1mL variable e e Protocol page 17 y of blood rive ul ai variable e e e Protocol J page 18 ee variable e e e Protocol K page 19 Buccal cells 2 variable e ProtocolL page 20 Saliva variable e Protocol M page 21 Hair follicles Bil variable e e Protocol N page 22 Milk ge variable e Protocol O Page 23 Resuspend Magnetic Beads well by vortexing before use on e gt N oo Prepare t
24. lect buccal cells by centrifugation at 5 000 x g for 10 minutes at 4 C C Collect mouthwash samples in 50 mL polypropylene conical test tubes and collect cells by centrifugation at 5 000 x g for 10 minutes at 4 C 2 Discard the supernatant For manual gDNA purification proceed to Step 2 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 3 proceed to Step 2 of Protocol A Instructions for genomic DNA purification from up to 106 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 11 20 Protocol M Instructions for genomic DNA purification from saliva Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described onpage 5 e Transfer the Tissue_gDNA Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Before starting e Prepare 1X TE buffer 10 mM Tris HCl pH 8 0 1 mM EDTA Step Procedure Protocol N Instructions for genomic DNA purification from hair follicles Note e When usi
25. nd Microtiter deep well 96 plates on page 11 Protocol K Instructions for genomic DNA purification from urogenital swabs Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA_Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Before starting e Prepare 1X TE buffer 10 mM Tris HCl pH 8 0 1 mM EDTA Protocol L Instructions for genomic DNA purification from human buccal cells Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA_Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Before starting e Prepare 1X TE buffer 10 mM Tris HCl pH 8 0 1 mM EDTA Step Procedure Step Procedure A Swab material collected using a shaft with cotton or brush tips should be placed into a long centrifuge tube 10 12 mL containing 500 uL TE buffer slightly vortex for 15 20 minutes and then remove the shafts with tips from tubes Collect cells by 1 centrifugation at 5 000 x g for 10 minutes at 4 C B If urogenital swabs were collected into LBC liquid based cytology medium collect cells for DNA pu
26. ng the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 e Transfer the Tissue_gDNA Flex protocol file to the KingFisher Flex or Tissue_gDNA_Duo protocol file to the Kingfisher Duo instrument as described on page 7 Before starting e Prepare 1X TE buffer 10 mM Tris HCl pH 8 0 1 mM EDTA 1 Collect cells from saliva diluted by equal volume of TE buffer by centrifugation at 5 000 x g for 10 minutes at room temperature Step Procedure 2 Discard the supernatant 1 Collect 8 10 hairs with follicles about 0 5 cm in length into a 2 0 mL microcentrifuge tube add 200 uL of Digestion Solution and resuspend For manual gDNA purification proceed to Step 2 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 3 proceed to Step 2 of Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 11 2 Add 20 uL of Proteinase K Solution and mix thoroughly by vortexing or pipetting to obtain a uniform suspension Incubate the samp
27. ponents of labelling guanidinium chloride Risk phrases 22 Harmful if swallowed 36 38 Irritating to eyes and skin Safety phrases 3 Keep in a cool place 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 x Proteinase K Xn Harmful Hazard determining components of labelling Proteinase Tritirachium album serine Risk phrases R42 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 administration 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 FTA is a trademark of GE Healthcare and its subsidiaries All other trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries 25
28. reparation a PBS 137 mM NaCl 2 7 mM KCl 10 mM NazHPOa 2 mM KH2PO pH 7 4 b 0 15 M NaCl solution c TE buffer 10 mM Tris HCl pH 8 0 1 mM EDTA For gram positive bacteria lysate preparation Gram positive bacteria lysis buffer 20 mM Tris HCI pH 8 0 2 mM EDTA 1 2 Triton X 100 add lysozyme to 20 mg mL immediately before use For yeast lysate preparation Yeast lysis buffer 5 mg mL Zymolyase 20T 1 M sorbitol 0 1 M EDTA For red blood cells lysis RBC buffer 10X buffer 1 68 M NH4Cl 2 mM EDTA STARTING MATERIAL HANDLING AND STORAGE To minimize DNA degradation avoid repeated freeze thaw cycles of the samples and perform extractions from fresh material or material that has been immediately frozen and stored at 20 C or 70 C Appropriate sample storage is essential for reproducibility and high DNA yields Yields of DNA may vary depending on sample age type of sample and storage conditions Yield of DNA purified from different types of tissue depends on efficiency of homogenization when grinding tissues tails insects or skin samples in liquid nitrogen using a mortar and pestle Disruption of tissue or rodent tail samples into small pieces using knives scissors or homogenizers results in 2 5 times reduced yield of DNA in comparison with grinding in liquid nitrogen For qualitative and quantitative DNA purification incubation of tissue lysates with Proteinase K up to 1 or 2 hours is sufficient Extension of the hy
29. rification from 2 mL of medium by centrifugation at 5 000 x g for 10 minutes at 4 C 2 Discard the supernatant For manual gDNA purification proceed to Step 2 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 3 proceed to Step 2 of Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 11 There are several buccal cell collection methods swabs brushes mouthwash and treated cards such as FTA cards A Swab samples obtained using plastic shafts and cotton tips should be immediately placed in a long centrifuge tube 10 12 mL containing 500 uL TE buffer thoroughly vortexed for 5 minutes and the swabs removed Collect cells by centrifugation at 5 000 x g for 10 minutes at 4 C 1 B If the swab material will not be used immediately allow it to dry and then place in a sealed plastic tube Before DNA purification place plastic shaft with cotton or brush tips into a long centrifuge tube 10 12 mL containing 500 uL TE buffer vortex gently for 10 15 minutes and then remove the shafts with tips from tubes Col
30. s the user and that nuclease free water is pipetted into the correct wells Elution strip Content Reagent volume per well KingFisher Duo elution strip Elution Buffer 150 uL 7 Place a Thermo Scientific KingFisher Duo 12 tip comb into row H on the DNA plate oo Switch on the KingFisher Duo instrument Start the Tissue_DNA_Duo protocol and load the plate and Elution Strip according to the KingFisher display Ensure that the Elution Strip is placed in the correct direction into the elution block and that the perforated end is facing toward the user The program will start after all plates have been loaded so After the run is completed remove the plate and 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 sterile tube and close immediately Store on ice for immediate use in downstream applications or store at 20 C Protocol D Instructions for genomic DNA purification from up to 20 mg tissue rodent tail and insects using KingFisher Duo and Microtiter deep well 96 plates Note e When using the MagJET Genomic DNA Kit for the first time prepare working solutions of Wash Buffer 1 and Wash Buffer 2 as described on page 5 Transfer the Tissue_gDNA_Duo protocol file to the KingFisher Duo as described on page 7 Ensure you are using the KingFisher Duo 12 pin magnet head and heating block
31. sample without any preservation reagents vortex 5 10 seconds and add 20 uL of Proteinase K solution mix by vortexing and incubate for 30 min at 56 C For manual gDNA purification transfer lysate to a tube prefilled with 400 uL of isopropanol and 25 uL of magnetic beads suspension resuspended well by vortexing and proceed to Step 5 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 For automated purification using KingFisher Flex 96 instruments transfer lysate to Sample plate prefilled with 400 uL of isopropanol and 25 uL of magnetic beads suspension and proceed to proceed to Step 3 of Protocol A Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 Note Skip treatment with RNase A step described on Step 5 For automated purification using KingFisher Duo instruments transfer lysate to Sample row prefilled with 400 uL of isopropanol and 25 uL of magnetic beads suspension according to Step 5 of Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 11 TROUBLESHOOTING Problem Possible cause and solution Excess sample used during lysate preparation Reduce the amount of starting material Do not use more tissue or cells than indicated in lysis protocols
32. tion from up to 10 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 10 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 plates on page 11 Step Procedure Harvest up to 109 bacterial cells 1 mL of overnight culture in a 1 5 or 2 mL i microcentrifuge tube by centrifugation for 10 min at 5 000 x g Discard the supernatant Resuspend the pellet in 40 uL of 0 15 M NaCl solution add 200 uL of Digestion Solution and 20 uL of Proteinase K Solution Mix the cell lysate thoroughly by vortexing or 2 pipetting to obtain a uniform suspension Incubate the sample at 56 C while vortexing occasionally or use a shaking water bath rocking platform or thermomixer until the cells are completely lysed 25 30 minutes For manual gDNA purification proceed to Step 4 of Protocol E Instructions for manual genomic DNA purification from up to 20 mg tissue rodent tail and insects on page 14 For automated purification using KingFisher Flex 96 or KingFisher Duo instruments 3 proceed to Step 3 of Protocol A Instructions for genomic DNA purification from up to 106 cultured mammalian cells using KingFisher Flex 96 and Microtiter deep well 96 plates on page 8 or Protocol C Instructions for genomic DNA purification from up to 106 cultured mammalian cells using KingFisher Duo and Microtiter deep well 96 p
33. tions for genomic DNA purification from blood IeUCOCYteS esceseeesseseeseeesteeeteeteeeeeees 17 Protocol J Instructions for genomic DNA purification from urine Sediment ccsceeecesseseesesesteesteeeeeees 18 Protocol K Instructions for genomic DNA purification from urogenital swabs cceceseeseeseeteeeteeteeeeeees 19 Protocol L Instructions for genomic DNA purification from human buccal cells cceseeseesesteeeeeteeeeeees 20 Protocol M Instructions for genomic DNA purification from saliva Protocol N Instructions for genomic DNA purification from hair follicles ceccecceseeeesesteseeteeteeeeteeeeeeeeees 22 Protocol O Instructions for genomic DNA purification from milk cecceeeceseesesesteseesesteseeeseeseesesneeeeeeeeenees 23 TROUBLESHOOTING sissisota niii aeiee lal Urea 24 SAFETY INFORMATION asiscncacraanaaccnuiienc ease yacaiinadieneniinaninains 25 COMPONENTS OF THE KIT MagJET Genomic DNA Kit Mines ace Proteinase K 2x 1 2 mL 8 x 1 2 mL Digestion Solution for MagJET gDNA Kit 22 mL 90 mL Lysis Buffer for MagJET gDNA Kit 35 mL 135 mL MagJET Magnetic Beads 2x1 4mL 10 6 mL RNase A 2x 1 2 mL 8 x 1 2 mL Wash Buffer 1 conc for MagJET gDNA Kit 25 mL 2 x 50 mL Wash Buffer 2 conc for MagJET gDNA Kit 50 mL 4x 50 mL Elution Buffer 30 mL 2 x 30 mL STORAGE Proteinase K solution is stable at room temperature as long as the vial remains sealed
34. tube measurement Purified DNA contains residual ethanol If residual ethanol is present in eluates of purified DNA include additional ier drying step 4 5 minutes at room temperature before the elution step into Inhibition of Fee dJownstreani DNA purification program of magnetic particle processors The instructions can be found in the Bindlt Software for KingFisher Instruments version 3 2 Enzymatic reactions User Manual Purified DNA contains residual salt Use the correct order of the Wash Buffers Always wash the magnetic beads with Wash Buffer 1 first and then proceed with Wash Buffer 2 Garryover of Carryover of MB in the eluted DNA will not affect downstream applications the magnetic beads MB To remove the carryover MB from eluted DNA simply magnetize the MB in the elution and carefully transfer to a new tube or plate 24 SAFETY INFORMATION x Lysis Buffer for MagJET 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 x Wash Buffer 1 conc for MagJET gDNA Kit Xn Harmful Hazard determining com
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