Troubleshooting Guide Contents
Contents
1. ethanol and pellet by pipetting up and down 5 times or shaking to mix well Place the plate onto a magnetic separation device to magnetize the MagSi DNA particles for 3 min Remove and discard the cleared supernatant Aspirate and discard the cleared supernatant Remove the plate from the magnetic separation device Add 500ul of 70 ethanol into the plate and pellet by pipetting up and down 5 times or 17 18 19 20 21 22 23 24 shaking to mix well Place the plate onto a magnetic separation device to magnetize the MagSi DNA particles for 3 min Remove and discard the cleared supernatant Remove and discard the cleared supernatant Do not remove the plate from the magnetic separation device Add 500ul Elution Buffer into the plate incubate for 30 seconds Remove and discard the cleared supernatant Remove the Plate from magnetic separation device Add 50 100ul Elution Buffer preheated at 70 C to elute DNA from the magnetic particles Resuspend MagSi DNA particles by pipetting up and down 20 30 times or shaking to mix well Incubate 5 minutes at 700C or 15 minutes at room temperature Place the plate onto magnetic separation device to magnetize the Mag Bead particles Wait 7 10 minutes or until magnetic beads are cleared from the solution Transfer the cleared supernatant containing purified DNA to a new 500ul microplate suplied Seal the plate with sealing film store at 20 C2. Product Number Purification Times 500 ul Micro Plate 1 2ml Process Plate 8 strip caps MagSi DNA Particles Buffer TL Buffer MSL Buffer MP Proteinase K RNase A Elution Buffer Instruction Booklet 1x96 Preps 1 1 10x 8 2 2 ml 20 ml 20 ml 30 ml 2 ml 550 ul 100 ml M6229 00 M6229 01 M6229 02 l 4x96 Preps 20 x 96 Preps 4 4 35x8 9 ml 90 ml 90 ml 100 ml 8 ml 2 1 ml 2 x 200 ml 1 20 20 170 x 8 45ml 2 x 180 ml 2 x 180 ml 8 x 100 ml 32 mt 10 mt 8 x 250 ml 1 E Z 96 MagSi DNA Tissue DNA Kit for Cultured cells This protocol is designed for the rapid isolation of up to 10 ug genomic DNA from up to 1 x 10 cultured cells 1 Prepare a RNase PBS working solution by mixing 5 ul RNase A with 100ul PBS for each sample For each 96 sets of sample prepare the Rnase PBS stock working solution by mix 0 5 mL RNase with 10 mL PBS 2 Prepare the cell suspension 2a Frozen cell samples should be thawed before starting this protocol Pellet the cells by centrifugation wash the cells with PBS and resuspend cells with 100ul cold 4 C PBS RNase Proceed with step 2 of this protocol 2b For cells grown in suspension pellet 1 x 10 cells by spinning at 1200 x g Discard the supernatant and wash the cells once with PBS Resuspend cells with 100ul cold 4 C PBS RNase 2c For cells grown in a monolayer harvest the cell by either using a trypsin treatment or scrape with rubber
3. genomic DNA from mouse tail snips blood buffy coat serum and plasma The kit allows single or multiple simultaneous processing of samples There is no need for phenol chloroform extractions and time consuming steps such as precipitation with isopropanol or ethanol are eliminated The Kit allows single or multiple simultaneous processing of samples in under 1 hours DNA purified using The E Z 96 Mag Binds Tissue DNA Kit is ready for applications such as PCR Southern blotting and restriction digestion Overview If using the E Z 96 Mag Binds Tissue DNA Kit for the first time please read this booklet in its entirety to become familiar with the procedures Tissue samples lysed in a specially formulated buffer The binding conditions are adjusted so that genomic DNA will selectively bind to the Mag Binds particles Two rapid wash steps remove trace contaminants such as residual polysaccharides and pure DNA is eluted in Elution Buffer Purified DNA can be directly used in downstream applications without the need for further purification Storage and Stability Most components of the E Z 96 Mag Bind Tissue DNA Kit except RNase A Proteinase K are stable for at least 24 months from date of purchase when stored at 22 25 C MagSi DNA Particles Solution should be stored at 4 C for long term use Store RNase A at 20 C Proteinase K should be stored at 15 25 C During shipment or storage in cool ambient conditions precipitates may f
4. Troubleshooting Guide Problem Likely Cause Suggestions 12 Low DNA yield Problem downstream applications in Incomplete resuspension of magnetic particle Frozen Tissue samples not mixed properly after thawing Loss the Mag Bind particle during operation DNA remains bound to Mag Bind Particles Ethanol carry over Resuspend the magnetic particles by vortexing before use Thaw the frozen Tissue at room temperature an d gently mix the Tissue by inverting Carefully avoid remove the Mag Bind particles during aspiration Increase elution volume and incubate at 650C for 5 min elution Pipet up and down for 50 100 times Dry the Mag Bind particle before elution Contents Introduction Overview Storage and Stability Kit Contents Before Starting E Z 96 Mag Binds Tissue Protocol 0 00 cece eee tenes E Z 96 Mag Binds Cultured Cell Protocol 0 0 0 cee eee neue E Z 96 Mag Binds Mouse Tail Protocol 020 cece eens E Z 96 Mag Binds Baccul Swabs Protocol 02 2 ee eee Troubleshooting Guide E Z 96 MagSi DNA Tissue DNA Kit for Buccal Swabs The following protocol is designed for isolating DNA from buccal swabs with magnetic beads 10 10 Place the swab into each well of 96 well plate no provided Prepare a Proteinase K Buffer TL working solution by mixing 20 ul Proteinas
5. and place into each well of 1 2ml process plate Make a chart to record the position of each sample 2 Prepare an Proteinase K Buffer TL working solution by mixing 20 ul Proteinase K with 130 ul Buffer TL for each sample For each 96 sets of samples prepare the Protease TL stock working solution by mix 2 0 mL protease with 13 mL Buffer TL Pipet 130 ul protease TL working solution into each well Seal the plate properly using the caps supplied 3 Mix the samples by inverting the plate 3 5 times Briefly spin the plate at 2 500 3 000 x g to collect any residue solution from the caps It is very important that samples are completely submerged in the solution 4 Incubate at 56 C overnight or until the samples are completely lysed The lysate should be clear and viscous after digestion is complete Mix occasionally during the incubation by rotating the plate gently Make sure the samples are completely lysed 5 Shake or vortex the plate vigorously from side to side Do not shake up and down to avoid leaking around the caps Ensure the lysate is completely homogenous after shaking If a gelatinous mass is visible further digestion is required Introduction The E Z 96 Mag Bind Tissue DNA Kit provides a rapid and easy method for the isolation of genomic DNA for consistent PCR and Southern analysis Up to 10 mg tissue or up to 1 cm sections of mouse tail can be readily processed in one time The method can also be used for preparation of
6. ase TL working solution into each well Seal the plate properly using the caps supplied Mix the samples by inverting the plate Briefly spin the plate at 2 500 3 000 x g to collect any residue solution from the caps It is very important that samples are completely submerged in the solution If the protease TL solution does not completely cover the sample increase the sample volume to 200ul Additional reagent can be purchased separately Incubate at 56 C over night or until the samples are completely lysed The lysate should be clear and viscous after digestion is complete Mix occasionally during the incubation by rotating the plate gently Make sure the samples are completely lysed Shake or vortex the plate vigorously from side to side Do not shake up and down to avoid leaking around the caps Hold the caps to ensure the plate is sealed properly Ensure the lysate is completely homogenous after shaking If a gelatinous mass is visible further digestion is required Optional For isolation of RNA Free genomic DNA spin briefly to collect any drops and add 5ul RNase A solution 20mg mL to each sample and incubate 10 20 minutes to remove the RNA Spin at 3 000 5 000 x g for 10 minutes at room temperature to remove undigested particles Transfer 150ul of the supernatant into each well of Process Plate Following step 7 24 on page 6 7 10 11 12 13 14 15 16 Optional Certain tissues such as liver ha
7. e K with 250ul Buffer TL for each sample For each 96 sets of sample prepare the Protease TL stock working solution by mix 2 0mL protease with 25mL Buffer TL Pipet 250 ul protease TL working solution into each well Seal the plate properly Incubate at 56 C for 1 hours Mix occasionally during the incubation by rotating the plate gently Make sure the samples are completely lysed Spin at 3 000 5 000 x g for 1 minutes at room temperature Transfer 150 ul of the Sample into each well of Plate provided Add 150ul Buffer MSL 20 ul MagSi gt DNA Paritcles and 220 ul absolute ethanol to the lysate Mix the sample by pipetting up and down 20 30 times to mix well or shaking to mix well Note Buffer MSL MagSi DNA Particles and absolute ethanol can be premixed The mixture can be stored at room temperature for 3 hours Place the Plate on a magnetic separation device MSD 01 to magnetize the MagSi DNA particles for 3 min The liquid should be cleared after all the magnetic beads are pelleted on the corner of the each well adjecent to the magnet Aspirate and discard the cleared supernatant Remove the Plate containing the MagSi DNA particles from the magnetic separation device Add 500ul Buffer MP diluted with ethanol into each well Note Buffer MP must be diluted with absolute ethanol 96 100 before use in this protocol Resuspend MagSi DNA particles pellet by pipetting up and down 20 times or shaking to mix well Kit Contents
8. orm in Buffer MSL and Buffer TL Dissolve such deposits by warming the solution at 37 C and gently shaking 11 12 13 14 15 16 17 18 19 20 21 22 23 Place the plate onto a magnetic separation device to magnetize the MagSi DNA particles for 3 min Remove and discard the cleared supernatant Remove the plate from the magnetic separation device Add 500ul of 70 ethanol and pellet by pipetting up and down 5 times or shaking to mix well Place the plate onto a magnetic separation device to magnetize the MagSi DNA particles for 3 min Remove and discard the cleared supernatant Aspirate and discard the cleared supernatant Remove the plate from the magnetic separation device Add 500ul of 70 ethanol into the plate and pellet by pipetting up and down 5 times or shaking to mix well Place the plate onto a magnetic separation device to magnetize the MagSi DNA particles for 3 min Remove and discard the cleared supernatant Remove and discard the cleared supernatant Do not remove the plate from the magnetic separation device Add 500ul Elution Buffer into the plate incubate for 30 seconds Remove and discard the cleared supernatant Remove the Plate from magnetic separation device Add 50 100ul Elution Buffer preheated at 70 C to elute DNA from the magnetic particles Resuspend MagSi DNA particles by pipetting up and down 20 30 times or shaking to mix Incubate 5 minutes at 700C or 15 min
9. policemen Wash cells twice and resuspend the cells with 100ul cold 4 C PBS RNase 3 Transfer the suspended cells into each well of Process plate Make a chart to record the position of each sample 4 Add 20 ul Proteinase K into each well of process plate mix well by shaking incubate at room temperature for 10 minutes 5 Add 100 ul Buffer MSL into each well of Process plate Seal the plate properly using the caps supplied Incubate at 65 C for 15 min 6 Add 20 ul MagSi DNA Particles and 160ul absolute ethanol into each well of process plate Mix the sample by pipetting up and down 20 30 times to mix well or shaking to mix well 7 Following steps 8 24 at pate 6 7 E Z 96 Mag Bind Tissue DNA Protocol for Tissues Materials to be provided by user Laboratory centrifuge capable of 3 000 5 000 x g equipped with swinging bucket rotor Adapter for deep well microplate Waterbath equilibrated to 56 C and 65 C Equilibrated sterile dH O water or Elution Buffer at 65 C Absolute 96 100 ethanol Multichannel pipet with tips E Z 96 Magnetic Separation Strand Cat MSD 01 OPTIONAL Although no mechanical homogenization of tissue is necessary pulverizing the samples in liquid nitrogen will improve lysis and reduce incubation time Once the liquid nitrogen has evaporated transfer the powdered tissue to a clean Racked Microtubes Add 130 ul Buffer TL Proteinase K and proceed to step 3 below 1 Mince 5 10 mg tissues
10. utes at room temperature Place the plate onto magnetic separation device to magnetize the Mag Bead particles Wait 7 10 minutes or until magnetic beads are cleared from the solution Transfer the cleared supernatant containing purified DNA to anew 500ul microplate suplied Seal the plate with sealing film store at 20 C 11 Before Starting Please read this booklet thoroughly to become familiar with the E Z 96 MagSi DNA Tissue DNA Kit procedures Equilibrate Elution Buffer or sterile dH O water or 10 mM Tris pH 9 0 at 65 C Dilute Buffer MP with absolute ethanol as follows and store at room temperature M6229 00 Add 75 ml absolute 96 100 ethanol M6229 01 Add 150 ml absolute 96 100 ethanol M6229 02 Add 150 ml absolute 96 100 ethanol to each bottle E Z 96 MagSi DNA Tissue DNA Kit for Mouse Tail Snips Snip one piece of mouse tail 0 1 0 2 cm in length place into a new Process plate If necessary cauterize the wound to stop bleeding Note Mice should not be older that 6 weeks since lysis will be more difficult resulting in suboptimal DNA yields If possible obtain tail biopsy at 2 4 weeks and freeze samples at 70 C until DNA is extracted Prepare a Proteinase K Buffer TL working solution by mixing 20 ul Proteinase K with 180uI Buffer TL for each sample For each 96 sets of sample prepare the Protease TL stock working solution by mix 2 0mL protease with 18mL Buffer TL Pipet 200 ul prote
11. ve high levels of RNA which will be co purified with DNA using this kit While it will not interfere with PCR the RNA may be removed at this point For isolation of RNA Free genomic DNA spin briefly to collect any drops and add 5ul RNase A solution 20mg mL to each sample and incubate 10 20 minutes to remove the RNA Add 150 ul Buffer MSL 20 ul MagSic DNA Paritcles and 220 ul absolute ethanol to the lysate Mix the sample by pipetting up and down 20 30 times to mix well or shaking to mix well Note Buffer MSL MagSi DNA Particles and absolute ethanol can be premixed The mixture can be stored at room temperature for 3 hours Place the Plate on a magnetic separation device MSD 01 to magnetize the MagSi DNA particles for 3 min The liquid should be cleared after all the magnetic beads are pelleted on the corner of the each well adjecent to the magnet Aspirate and discard the cleared supernatant Remove the Plate containing the MagSi DNA particles from the magnetic separation device Add 500ul Buffer MP diluted with ethanol into each well Note Buffer MP must be diluted with absolute ethanol 96 100 before use in this protocol Resuspend MagSi DNA particles pellet by pipetting up and down 20 times or shaking to mix well Place the plate onto a magnetic separation device to magnetize the MagSi DNA particles for 3 min Remove and discard the cleared supernatant Remove the plate from the magnetic separation device Add 500ul of 70
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