Simultaneous purification of genomic DNA and total
Contents
1. 7 Open the tube and incubate for 5 min at 37 C using a shaker incubator for evaporation of residual alcohol After incubation set the temperature of the shaker incubator to 45 C for use in step 10 8 Add 150 pl Buffer TM1 and resuspend the pellet by vortexing for 20 s 9 Add 130 pl RNase free water to the resuspended pellet Then add 20 pl proteinase K and mix by vortexing for 5 s Note Do not mix Buffer TM1 and proteinase K together before adding them to the sample PAXgene Tissue AllPrep DNA RNA miRNA from sections of PFPE tissue PX10 Feb 13 page 3 of 7 10 11 12 13 Incubate for 15 min at 45 C using a shaker incubator at 1400 rpm After incubation centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the inside of the tube lid Set the temperature of the shaker incubator to 65 C for use in step 21 Note For purification of DNA and RNA from fibrous tissue e g skin heart or skeletal muscle aorta incubate for 2 h at 45 C Add 200 pl Buffer TM1 resuspend the pellet by vortexing for 5 s and centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the inside of the tube lid Pipet the sample including any precipitate that may have formed into a PAXgene DNA spin column placed in a 2 ml processing tube and centrifuge for 1 min at 6000 x g If the lysate has not completely passed through the membrane after centrifugation centrifuge again at a higher speed until the PAXgene DNA spin colu2. Note Buffer TD3 is supplied as a concentrate Ensure that ethanol is added to Buffer TD3 before use see Things to do before starting page 3 Add 25 ul proteinase K to 50 pl Buffer TD3 in a 1 5 ml microcentrifuge tube Mix by gently flicking the tube and centrifuge briefly to collect residual liquid from the sides of the tube For example if processing 10 samples add 250 ul proteinase K to 500 ul DNA Buffer TD3 Use the 1 5 ml microcentrifuge tubes supplied with the kit Pipet the proteinase K incubation mix 75 pl directly onto the PAXgene DNA spin column and incubate for 30 min at ambient temperature 20 30 C Pipet 350 ul Buffer TD3 into the PAXgene Tissue DNA spin column and centrifuge for 1 min at 6000 x g Place the spin column in a new 2 ml processing tube and discard the old processing tube containing flow through Pipet 500 pl Buffer TD4 into the PAXgene DNA spin column and centrifuge for 1 min at 6000 x g Place the spin column in a new 2 ml processing tube and discard the old processing tube containing flow through Centrifuge for 3 min at maximum speed but not to exceed 20 000 xg to dry the membrane completely This step is necessary since ethanol carryover into the eluate may interfere with some downstream applications Discard the processing tube containing the flow through Place the PAXgene DNA spin column in a 1 5 ml microcentrifuge tube and pipet 20 200 pl Buffer TD5 directly onto the P
3. Sterile aerosol barrier RNase free pipet tips e Graduated cylindert e Variable speed microcentrifuge capable of attaining 1000 8000 x g and equipped with a rotor for 2 ml microcentrifuge tubes Ensure that equipment has been checked and calibrated according to the manufacturer s recommendations P P r P ti t For the addition of isopropanol to Buffer TM2 and ethanol to Buffer TM3 concentrate and for preparation of 80 ethanol A QIAGEN BD Company www PreAnalytiX com Shaker incubator capable of incubating at 45 C and 65 C and shaking at 400 rpm not exceeding 1400 rpm e g Eppendorf Thermomixer Compact wwww ependorf com t or equivalent Vortex mixer Scalpel Crushed ice Starting material Starting material for genomic DNA and total RNA including miRNA purification should be 1 to 5 sections of PFPE PAXgene Tissue fixed paraffin embedded tissue see the PAXgene Tissue Container and the PAXgene Tissue FIX Container 50 ml Product Circular for information about tissue fixation stabilization and paraffin embedding Each section should have a thickness of 5 10 um and a tissue surface area of up to 225 mm Thicker sections may result in lower nucleic acids yields Important points before starting If working with RNA for the first time see Appendix A General Remarks on Handling RNA in the PAXgene Tissue miRNA Kit Handbook Ensure that the kit boxes are intact and undamaged and tha
4. elute the RNA Note It is important to wet the entire membrane with Buffer TM4 to achieve maximum elution efficiency Smaller volumes of Buffer TM4 can be used to obtain a higher total RNA concentration but this will influence the overall yield The dead volume of the PAXgene RNA MinElute spin column is 2 ul elution with 14 ul Buffer TM4 results in an eluate with a volume of 12 ul Incubate the eluate for 5 min at 65 C in the shaker incubator from step 10 without shaking After incubation chill immediately on ice Note This incubation at 65 C denatures the RNA for downstream applications Do not exceed the incubation time or temperature If the RNA samples will not be used immediately store at 15 to 30 C or 70 C Since the RNA remains denatured after repeated freezing and thawing it is not necessary to repeat the incubation at 65 C Note For quantification in Tris buffer use the relationship Ayo 1 gt 44 ug ml See Appendix A General Remarks on Handling RNA in the PAXgene Tissue miRNA Kit Handbook PAXgene Tissue AllPrep DNA RNA miRNA from sections of PFPE tissue PX10 Feb 13 page 5 of 7 Genomic DNA purification 23 24 25 26 27 28 29 Pipet 350 ul Buffer TD3 into the PAXgene Tissue DNA spin column from step 13 and centrifuge for 1 min at 6000 x g Place the spin column in a new 2 ml processing tube and discard the old processing tube containing flow through
5. 0 x g Place the spin column in a new 2 ml processing tube and discard the old processing tube containing flow through Note Buffer TM2 is supplied as a concentrate Ensure that isopropanol is added to Buffer TM2 before use see Things to do before starting page 3 Flow through contains Buffer TM1 or Buffer TM2 and is therefore not compatible with bleach PAXgene Tissue AllPrep DNA RNA miRNA from sections of PFPE tissue PX10 Feb 13 page 4 of 7 17 18 19 20 21 22 Pipet 500 pl Buffer TM3 into the PAXgene RNA MinElute spin column and centrifuge for 20 s at 8000 x g Place the spin column in a new 2 ml processing tube and discard the old processing tube containing flow through Note Buffer TM3 is supplied as a concentrate Ensure that ethanol is added to Buffer TM3 before use see Things to do before starting page 3 Pipet 500 pl 80 ethanol into the PAXgene RNA MinElute spin column and centrifuge for 2 min at 8000 x g Discard the processing tube containing the flow through and place the PAXgene RNA MinElute spin column in a new 2 ml processing tube Open the cap of the spin column and centrifuge for 5 min at maximum speed Discard the processing tube containing the flow through Place the PAXgene RNA MinElute spin column in a 1 5 ml microcentrifuge tube and pipet 14 40 ul Buffer TM4 directly onto the PAXgene RNA MinElute spin column membrane Centrifuge for 1 min at maximum speed to
6. AXgene DNA spin column membrane Centrifuge for 1 min at maximum speed but not to exceed 20 000 x g to elute the DNA Incubating the PAXgene DNA spin column loaded with Buffer TD5 for 5 min at room temperature 15 25 C before centrifugation generally increases DNA yield Flow through contains Buffer TD2 or Buffer TD3 and is therefore not compatible with bleach PAXgene Tissue AllPrep DNA RNA miRNA from sections of PFPE tissue PX10 Feb 13 page 6 of 7 For up to date licensing information and product specific disclaimers see the respective PreAnalytix or QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www qiagen com or can be requested from QIAGEN Technical Services or your local distributor Safety data sheets SDS for any QIAGEN or PreAnalytiX product can be downloaded from www giagen com safety Trademarks PAXgene PreAnalytix PreAnalytiX GmbH QIAGEN AllPrep MinElute QIAGEN Group Eppendorf Eppendorf AG PX10 February 13 2013 PreAnalytiX all rights reserved A PreAnalytiX A QIAGEN BD Company www PreAnalytiX com
7. PreAnalytiX Supplementary Protocol PAXgene Tissue AllPrep DNA RNA miRNA Simultaneous purification of genomic DNA and total RNA including miRNA from sections of PAXgene Tissue fixed paraffin embedded PFPE tissue This protocol is designed for using the PAXgene Tissue miRNA and the PAXgene Tissue DNA Kit for simultaneous purification of genomic DNA and total RNA including miRNA from the same sections of PFPE tissue Before beginning the tissue sample must be fixed and stabilized in one of the PAXgene Tissue Containers dehydrated and embedded in paraffin IMPORTANT Please read the PAXgene Tissue miRNA Kit Handbook and the PAXgene Tissue DNA Kit Handbook paying careful attention to the Safety Information and Important Notes sections before beginning this procedure For research use only Not for use in diagnostics procedures No claim or representation is intended to provide information for the diagnosis prevention or treatment of a disease Equipment and reagents When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier e Xylene e Staining dishes e Horizontal working plate e Ethanol 96 100 purity grade p a e Isopropanol e 14 3 M B mercaptoethanol B ME commercially available solutions are usually 14 3 M e Pipets 10 ul 1 ml e
8. mn is empty Place the PAXgene DNA spin column in a new 2 ml collection tube supplied and store at room temperature 15 25 C or at 4 C for up to 24 hours for later DNA purification in steps 23 29 Use the flow through for RNA purification in steps 14 22 Note Do not store the PAXgene DNA spin column at room temperature or at 4 C for more than 24 hours Do not freeze the column Total RNA including miRNA purification 14 15 16 Add 750 ul isopropanol to the flowthrough from step 13 Mix by vortexing for 5 s and centrifuge briefly 1 2 s at 500 1000 x g to remove drops from the inside of the tube lid Note The length of the centrifugation must not exceed 1 2 s as this may result in pelleting of nucleic acids and reduced yields of total RNA Note A precipitate may form after the addition of ethanol but this will not affect the PAXgene Tissue RNA procedure Pipet the sample including any precipitate that may have formed into a PAXgene RNA MinElute spin column red placed in a 2 ml processing tube and centrifuge for 1 min at 8000 x g Place the spin column in a new 2 ml processing tube and discard the old processing tube containing flow through If the lysate has not completely passed through the membrane after centrifugation centrifuge again at a higher speed until the PAXgene RNA MinElute spin column is empty Pipet 700 pl Buffer TM2 into the PAXgene RNA MinElute spin column Centrifuge for 20 s at 800
9. st be added to Buffer TM before use Add 10 ul B ME per 1 ml Buffer TM1 Dispense in a fume hood and wear appropriate protective clothing Buffer TM1 containing B ME can be stored at room temperature 15 25 C for up to 1 month e Buffers TM2 TM3 TD3 and TD4 are supplied as concentrates Before using for the first time add isopropanol or ethanol 96 100 purity grade p a as indicated on the bottle to obtain a working solution Procedure 1 Using a microtome generate up to 5 tissue sections of 5 10 um thickness from the PFPE tissue Note If the sample surface has been exposed to air discard the first 2 or 3 sections 2 Place sections in a 1 5 ml microcentrifuge tube 3 Add 650 pl xylene to the sample Vortex vigorously for 20 s and incubate for 3 min on the benchtop at 15 25 C 4 Add 650 pl ethanol 96 100 purity grade p a and mix by vortexing for 20 s 5 Centrifuge at maximum speed for 5 min but do not exceed 20 000 x g To prevent damage to processing tubes do not exceed 20 000 x g 6 Remove the supernatant by pipetting Do not remove any of the pellet Note In some cases the pellet may be loose Remove the supernatant carefully Note It is essential to remove all residual alcohol from the pellet Note The pellet might contain residual paraffin however the paraffin will dissolve during digestion with proteinase K and will not affect the PAXgene Tissue AllPrep DNA RNA miRNA procedure
10. t buffers have not leaked Do not use a kit that has been damaged When using a pipet ensure that it is set to the correct volume and that liquid is carefully and completely aspirated and dispensed To avoid transferring samples to the wrong tube or spin column ensure that all tubes and spin columns are properly labeled Label the lid and the body of each tube For spin columns label the body of its processing tube Close each tube or spin column after liquid is transferred to it Spillages of samples and buffers during the procedure may reduce the yield and purity of RNA Unless otherwise indicated all steps of this protocol including centrifugation steps should be carried out at room temperature 15 25 C Ensure that equipment has been checked and calibrated according to the manufacturer s recommendations t This is not a complete list of suppliers and does not include many vendors of biological supplies PAXgene Tissue AllPrep DNA RNA miRNA from sections of PFPE tissue PX10 Feb 13 page 2 of 7 Things to do before starting e Before using the kit for the first time prepare 80 ethanol by mixing 24 ml ethanol 96 100 and 6 ml RNase free water supplied e Ashaker incubator is required in steps 7 10 and 21 Set the temperature of the shaker incubator to 37 C e Buffer TM1 and Buffer TM2 may form a precipitate upon storage If necessary warm to 37 C to dissolve e Mercaptoethanol B ME mu
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