Troubleshooting Guide
Contents
1. Absorbance x 50 x Dilution Factor ug ml The ratio of absorbance absorbance is an indication of nucleic acid purity A value greater than 1 8 indicates gt 90 nucleic acid Alternatively quantity as well as quality can sometimes best be determined by agarose gel ethidium bromide electrophoresis by comparison to DNA samples of known concentrations E Z N A Mag Bind Forensic DNA Kit Magnetic Protocol Materials to be provided by user Centrifuge capable of 12 000 x g Nuclease free 15 ml centrifuge tube Water bath preset at 65 C Absolute 96 100 ethanol Magnetic separation strand 70 ethanol Transfer the forensic sample to a sterile microcentrifuge tube and add 180 ul Buffer TL 20 yl OB Protease Vortex to mix well making sure that the sample is completely immersed in the Buffer TL Incubate the sample for 1 3 hours at 55 C until lysis is complete If RNA free genomic DNA is required add 5 yl RNase A to each sample Remove the sample with disposable tweezers or transfer 200 ul the lysate supernatant to a new tube Add200 ul Buffer MLS 20 ul MagSi Particles and 300 ul absolute ethanol room temperature 96 100 to the lysate Vortexing Shaking for 2 minutes or pipetting up and down 20 30 times to mix well Note Buffer MSL MagSi Particles and absolute ethanol can be premixed Place the tube on a magnetic separation device suitable for 1 5 ml tube to magnetize the Mag Bind particles Remove and2. particles Remove and discard the cleared supernatant Leave the tube to air dry on the magnetic separation device for 5 minutes Remove any residue liquid from tube by pipetting Remove the tube from magnetic separation device Add 50 100ul Elution Buffer or water to elute DNA from the magnetic particles Resuspend Mag Bind particles by vortexing shaking or pipetting up and down 20 times Incubate 5 10 minutes at room temperature Repeating the mix by vortexing or pipetting up and down for 1 minutes Place the tube onto a magnetic separation device to magnetize the Mag Bead particles Transfer the cleared supernatant containing purified DNA to a new 1 5 ml tube
3. please read this booklet in its entirety to become familiar with the procedures Forensic samples lysed in a specially formulated buffer The binding conditions are adjusted so that genomic DNA will selectively bind to the Mag Binds 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 N A Mag Binds Forensic DNA Kit except RNase A and OB Protease are stable for at least 24 months from date of purchase when stored at 22 C 25 C Mag Bind Particles Solution B should be stored at 4 C for long term use Store RNase A at 20 C OB Protease store at 15 25 C During shipment or storage in cool ambient conditions precipitates may form in Buffer MBL Dissolve such deposits by warming the solution at 37 C and gently shaking 10 Add 50 100ul Elution Buffer or water to elute DNA from the magnetic particles 11 Resuspend Mag Bind particles by vortexing Incubate 5 10 minutes at room temperature 12 Centrifuge at 10 000 x g for 1 min and transfer the supernatant into a new tube Store Purified DNA at 20 C Yield and quality of DNA Determine the absorbance of an appropriate dilution 20 to 50 fold of the sample at 260 nm and then at 280 nm The DNA concentration is calculated as follows DNA concentration
4. Troubleshooting Guide Problem Cause Suggestions Low DNA yield Problems in downstream applications Incomplete disruption of starting material Poor lysis of tissue Loss the MagBeads particle during operation DNA remains bound to MagBeads Particles DNA washed off Salt carry over Ethanol carry over For both dry and fresh samples obtain a fine homogeneous powder before adding Buffer SP1 Decrease amount of starting material or increase amount of Buffers SP1 and SP2 Carefully avoid remove the MagBeads particles during aspiration Increase elution volume and incubate on column at 65 C for 5 min elution Dilute MGB Binding Buffer and SPM Wash Buffer by adding appropriate volume of absolute ethanol prior to use page 3 Wash Buffer must be at room temperature Dry the MagBeads particle before elution Contents Introduction 022008 OVERVIEW i cca dee eR ee EE eee eas Storage and Stability Kit Contents 0 ee ee eee eee Before Starting 0 008 E Z N A Mag Binds Forensic DNA Magnetic Protocol E Z N A Mag Binds Forensic DNA Spin Protocol Troubleshooting Guide E Z N A Mag Bind Forensic DNA Kit Spin Protocol Kit Contents The E Z N A Mag Binds Forensic DNA Kit May be proceed by Centrifugation if Product Number M6225 00 M6225 01 M6225 02 without magnetic strand
5. available The purity of genomic DNA isolated by Spin Protocol may be low than Magnetic protocol because some unsoluble particle Purifications Times 5 Preps 50 Preps 200 Preps precipitated with genomic DNA while only genomic DNA can be collected in MagSi Particles 120ul 1 2ml 4x1 1 ml Magnetic Protocol In most specimens genomic DNA isolated by this protocol can be used in PCR Southern blot Buffer TL 5 ml 20 ml 60 ml Buffer MSL 5 ml 20 ml 60 ml 1 Transfer the forensic sample to a sterile microcentrifuge tube and add Buffer MP Imi 20 ml 80 ml 180 ul Buffer TL 20 ul OB Protease Vortex to mix well making sure that the sample is completely immersed in the Buffer TL OB Protease 150ul 1 2 ml 4x 1 2 ml RNase A 12ul 120ul 440ul 2 Incubate the sample for at least 1 hours at 55 C until lysis is complete Elution Buffer iml 20 ml 60 ml 3 Remove the Forensic sample with disposable tweezers or Centrifuge at Instruction Booklet 1 1 1 15 000 x g for 5 min and transfer 200ul supernatant to a new tube 4 Add200 ul Buffer MLS 20 ul MagSi Particles and 300 ul absolute ethanol room temperature 96 100 to the lysate Vortexing Shaking for 2 minutes or pipetting up and down 20 30 times to mix well Before Starting e Please read this booklet thoroughly to become familiar with the E Z N A Mag Bind Forensic DNA Kit procedures Note Buffer MSL MagSi Particles and absolute ethanol can be premixed ae 7 Equilibrate Elution B
6. discard the cleared supernatant Tip To ensure that all traces of the medium are removed let the tube sit 2 min at room temperature and remove the remaining liquid by pipettor or invert the tube on paper Remove the tube containing the Mag Bind particles from the magnetic separation device Add 400ul Buffer MP3 diluted with ethanol into the tube Note Buffer MP3 must be diluted with absolute ethanol 96 100 before use in this protocol 10 11 12 13 14 15 16 17 18 19 20 21 Resuspend Mag Bind particles pellet by votexing shaking for 1 minutes or pipetting up and down 20 times Place the plate onto a magnetic separation device to magnetize the Mag Bind particles Remove and discard the cleared supernatant Remove the tube containing the Mag Bind particles from the magnetic separation device Add 500ul of 70 ethanol into the tube Resuspend Mag Bind particles pellet by vortexing shaking for 1 minute or pipetting up and down 20 times Place the plate onto a magnetic separation device to magnetize the Mag Bind particles Remove and discard the cleared supernatant Remove the tube containing the Mag Bind particles from the magnetic separation device Add 500ul of 70 ethanol into the tube Resuspend Mag Bind particles pellet by vortexing shaking for 1 minute or pipetting up and down 20 times Place the plate onto a magnetic separation device to magnetize the Mag Bind
7. uffer or sterile dH O water at 65 C 5 Centrifuge at 10 000 x g for 1 min Remove and discard the cleared supernatant o Dilute Buffer MP with absolute ethanol as follows and store at room temperature Tip To ensure that all traces of the solution are removed invert the tube on paper for 2 min M6225 00 Add 3 ml absolute 96 100 ethanol M6225 01 Add 30 ml absolute 96 100 ethanol 6 Add 400 ul of Buffer MP3 and vortex to resuspend Mag Bind Pellets M6225 02 Add 120 ml absolute 96 100 ethanol Note Buffer MP3 should not diluted with absolute ethanol before use in this spin protocol 7 Add 500 ul of 70 ethanol vortex to mix 8 Centrifuge at 10 000 x g for 1 min Remove and discard the cleared supernatant 9 Leave the tube to air dry on the magnetic separation device for 5 minutes Introduction The E Z N A Mag Binds Forensic DNA Kit allows rapid and reliable isolation of high quality genomic DNA from Forensic The Kit allows single or multiple simultaneous processing of samples in under 1 hours There is no need for phenol chloroform extraction and time consuming steps such as CsCl gradient ultracentrifugation and precipitation with isopropanol or ethanol are eliminated DNA purified using the E Z N A Mag Binds Forensic DNA Kit is ready for applications such as PCR Southern blotting and restriction digestion Overview If using the E Z N A Mag Binds Forensic DNA Kit for the first time
Download Pdf Manuals
Related Search