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CST Genomic DNA Purification Kits -- Tissues

1. 58 C 70 C 70 C Note The optimal incubation temperature for Genomed Protease is 58 C However to reach the catalytic temperature range as quickly as possible incubate Midiprep and Maxiprep samples at 70 C If incubation at 58 C is desired extend the incubation time to 20 minutes Add ethanol and mix by vortexing Reagent Miniprep Midiprep Maxiprep 96 100 Ethanol 200 uL 3mL 10 mL Note Mix the sample immediately to prevent precipitation of nucleic acids due to high local alcohol concentrations Proceed immediately to Purifying DNA next page Continued on next page 13 Purifying gDNA from Blood and Body Fluids Using Centrifugation Continued Purifying DNA 14 1 Assemble a JetQuick Spin Column with an appropriate receiver tube Spin Column Miniprep Midiprep Maxiprep Assembly 2mL Receiver Tube Lasrovided 50 mi 50 mL size in the kit Note For 50 mL tubes loosely attach a cap to allow ventilation during centrifugation Apply the sample from Step 5 previous page into the JetQuick Spin Column Note For Maxiprep starting blood volumes gt 5 mL load 15 mL blood lysate onto the JetQuick Maxi Spin column with two successive rounds of centrifugation Centrifuge samples using the following conditions Centrifugation Miniprep Midiprep Maxiprep Acceleration 10 000 xg 2 000 x g 2 000 x g
2. Optional To recover more DNA perform a second elution step in another sterile 1 5 mL microcentrifuge tube Centrifuge the column at 12 000 x g for 2 minutes at room temperature The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application Storing DNA To avoid repeated freezing and thawing of DNA Store the purified DNA at 4 C for immediate use or Aliquot the DNA and store at 20 C for long term storage 31 Purifying gDNA from Swabs Introduction The procedure for purifying genomic DNA from buccal nasal pharyngeal and vaginal swabs using JetQuick Blood amp Cell Culture DNA Miniprep Kits is described below To obtain high quality genomic DNA follow the guidelines recommended on page 4 Materials e 96 100 ethanol Needed e Swab Sample e Phosphate Buffered Saline PBS or equivalent saline buffer i e TBS see page 57 for ordering information e Sterile DNase free microcentrifuge tubes e Microcentrifuge capable of centrifuging at 212 000 x g e Water baths or heat blocks Components supplied with the JetQuick Blood amp Cell Culture DNA Miniprep Kit e Buffers K1 K2 and KX e Genomed Protease lyophilized powder see Before Starting page 5 e RNase A 20 mg mL e 10 mM Tris HCl pH 8 5 e Je
3. amp Cell Culture DNA Mini Midi and Maxiprep Kits are designed to purify gDNA from any type of body fluid e g amniotic fluid saliva sperm lymph and fresh or frozen whole blood which may be collected in the presence of anti coagulants such as EDTA or citrate To obtain high quality genomic DNA follow the guidelines recommended on page 4 For a procedure using Vacuum manifold see page 17 96 100 ethanol Sample for DNA isolation see page 10 Sterile DNase free microcentrifuge tubes Miniprep only Sterile DNase free 50 mL tubes Midiprep and Maxiprep Water baths or heat blocks Microcentrifuge capable of centrifuging 212 000 x 2 Centrifuge with a swing out rotor capable of centrifuging 2 000 5 000 x g Midiprep and Maxiprep only Optional Phosphate Buffered Saline PBS see page 57 or equivalent standard saline buffer for processing 1 mL Miniprep 20 mL Maxiprep or buffy coat samples Optional White Blood Cell Buffer see page 11 for processing 1 mL Miniprep or 20 mL Maxiprep samples Components supplied with the JetQuick Blood amp Cell Culture DNA Mini Midi and Maxiprep Kits Buffers K1 K2 and KX Genomed Protease lyophilized powder see page 5 RNase A 20 mg mL 10 mM Tris HCl pH 8 5 JetQuick Spin Columns JetQuick Receiver Tubes Miniprep Kits only Continued on next page Purifying gDNA from Blood and Body Fluids Using Centrifugation Continued Experimental Outline MiniPrep
4. concentration 1 5 mL Midiprep Standard no 12 modification 6 10 mL Maxiprep Standard no 12 modification 11 20 mL Maxiprep Requires lysate 11 concentration Blood from a healthy person contains on average 5 x 106 1x107 DNA containing lymphocytes per mL The values in the table are for nonnucleated blood e g human or mouse Nucleated starting blood e g bird volumes will be substantially lower e g 5 10 uL for Miniprep Scale up or down the volumes of all buffers and components proportionally if using a non standard starting volume e g standard starting volumes are 3 mL whole blood for Midiprep and 10 mL for Maxiprep For body fluids you may use the volumes indicated in the table above However if the body fluid sample is dilute or has few DNA containing cells concentrate the cells by centrifugation 12 000 x 2 for 30 seconds for Miniprep or 5 000 x 2 for 2 minutes for a Midiprep or Maxiprep Following centrifugation aspirate the supernatant and resuspend the cell pellet in 200 uL Mini 3 mL Midi or 10 mL Maxi PBS Continued on next page Purifying gDNA from Blood and Body Fluids Using Centrifugation Continued Buffy Coat Samples Preparing Concentrated Lysates Use no more than 1 2 x10 cells per buffy coat preparation Adjust the volume of buffy coat corresponding to this amount of cells with PBS TBS or another standard saline buffer to be provided by the user to
5. 70 C Midi and Maxi Harvest cells by adding fresh or frozen blood body fluid serum plasma or buffy coat to a clean sterile tube Starting Miniprep Midiprep Maxiprep Materials Starting Sample 200 pL 3mL 10 mL Volume Harvesting Tube 15mL 50 mL 50 mL Size Continued on next page Purifying gDNA from Blood and Body Fluids Using Vacuum Continued Preparing Lysate Continued Add the reagents in the order listed in the table below and mix thoroughly by vortexing or inverting the tube Do not add Genomed Protease directly to Buffer K1 First mix the suspended cells with the enzyme and then add Buffer K1 Reagent Miniprep Midiprep Maxiprep Operation SA 20 uL 300 uL 500 uL iie 10 pL 100 pL 300 uL Mix sample vortex vortex vortex Buffer K1 200 uL 3 mL 10 mL Mix sample vortex vortex vortex Incubate the sample for 10 minutes at the temperature indicated in the table below to degrade protein Incubation Miniprep Midiprep Maxiprep Temperature 58 C 70 C 70 C Note The optimal incubation temperature for Genomed Protease is 58 C However to reach the catalytic temperature range as quickly as possible incubate Midiprep and Maxiprep samples at 70 C If incubation at 58 C is desired extend the incubation time to 20 minutes Add ethanol and mix by vortexing Reagent Miniprep Midiprep Maxiprep 96 100 Ethanol 200
6. Miniprep JetQuick Blood amp Cell Culture DNA Miniprep Kit and analyzed by agarose gel electrophoresis on a 176 TAE gel Samples on the gel are Lanes 1 10 Lambda HindIII EcoRI Lane 2 DNA isolated from fresh EDTA blood Lane 3 DNA isolated from EDTA blood stored at 4 C for 2 days Lane 4 DNA isolated from frozen EDTA blood Lane 5 DNA isolated from fresh citrate blood Lane 6 DNA isolated from citrate blood stored at 4 C for 2 days Lane 7 DNA isolated from frozen citrate blood Lane 8 DNA isolated from fresh heparin blood Lane 9 DNA isolated from frozen heparin blood 127 3 45 5 7 6 3 10 T T ee ee dA M 23 Kb 2 Kb Continued on next page 52 Examples of Expected Results Continued Blood gDNA Midi and Maxiprep Tissue gDNA Miniprep Genomic DNA isolated from various samples was analyzed by agarose gel electrophoresis on a 0 8 TAE gel 500 ng Midiprep or 700 ng Maxiprep blood gDNA per sample from 7 Midiprep or 3 Maxiprep blood samples prepared in parallel with the JetQuick Blood amp Cell Culture DNA Midiprep or Maxiprep Kits Lambda DNA digested with EcoRI HindIII was used as a DNA size marker in lane 1 for both gels Midi Maxi 12345678 12 34 23 Kb Genomic DNA isolated from various tissue samples using a JetQuick Tissue DNA Miniprep Kit was analyzed by agarose gel electrophoresis on a 1 TAE gel Lane contents are as follows Lanes 1 8 Lambda Hindl
7. an overall volume of 3 mL Midiprep or 10 mL Maxiprep If you are using standard starting blood volumes of up to 200 pL for Miniprep 1 5 mL for Midiprep or 6 10 mL for Maxiprep see page 10 skip this section To purify gDNA from 300 uL to 1 mL blood samples with the JetQuick Blood Miniprep Kit or 11 to 20 mL blood samples with the JetQuick Blood Maxiprep Kit 1 Prepare WBC Buffer 10 mM Tris HCl pH 8 5 5 mM MgCl 320 mM Sucrose 196 Triton X 100 2 Place whole blood sample into a sterile harvesting tube Starting Materials Miniprep Maxiprep Starting Sample Volume 300uL 1mL 11 20 mL Harvesting Tube Size 2mL 50mL 3 Addanequal volume of WBC Buffer to the blood sample Mix thoroughly by inverting the tube several times 4 Centrifuge using the following conditions Centrifugation Miniprep Maxiprep Acceleration 12 000 x g 5 000 x g Duration 30 seconds 2 minutes Continued on next page 11 Purifying gDNA from Blood and Body Fluids Using Centrifugation Continued Preparing 5 Aspirate the supernatant with a pipette Do not disturb the Concentrated white blood cell pellet which appears light red Lysates 6 Resuspend the cell pellet in PBS or equivalent standard Continued saline buffer Mix by pulse vortexing Reagent Miniprep Maxiprep Volume PBS 180 uL 9 5 mL 7 Proceed immediately to Preparing Lysate below Preparing Prepare
8. compliance with established institutional guidelines and take the appropriate precautions wear a laboratory coat disposable gloves and eye protection when handling blood and tissue samples Since safety requirements for use and handling of blood and tissue samples may vary at individual institutions consult the health and safety guidelines and or officers at your institution When processing blood and tissue samples the eluates collected during wash steps contain biohazardous waste Dispose the eluate and collection tubes plates appropriately as biohazardous waste Buffers K1 KX T2 and TX contain guanidine hydrochloride Always wear a laboratory coat disposable gloves and eye protection when handling buffers Do not add bleach or acidic solutions directly to solutions containing guanidine hydrochloride or sample preparation waste as it forms reactive compounds and toxic gases when mixed with bleach or acids Continued on next page Milli O is a registered trademark of Millipore Corporation General Information Continued RNase A e RNA contamination inflates the nucleic acid content Digestion measured at 260 nm e RNase A digestion is performed during sample preparation to degrade RNA present in the sample and minimize RNA contamination in the purified DNA sample e RNase A is supplied with the kit and an optional RNase A digestion step is included during sample preparation protocols e If RNA content of the sa
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10. precipitated If any precipitated matter is present co transfer it into the spin column Centrifuge the sample for 1 minute at 10 000 x g Discard the flow through and re assemble the JetQuick Spin Column into the Receiver Tube Add 500 pL Buffer TX with ethanol see page 5 to the column Centrifuge column at room temperature at 10 000 x g for 1 minute Discard the flow through and re assemble the JetQuick Spin Column Add 500 pL Buffer T3 with ethanol page 5 to the column Centrifuge column at room temperature at 10 000 x g for 1 minute Discard the flow through and re assemble the JetQuick Spin Column using the same 2 mL Receiver Tube Centrifuge the column again at 12 000 x g for 1 minute at room temperature to clear the silica membrane of any residual liquid Discard the 2 mL Receiver Tube Place the JetQuick Spin Column into a sterile 1 5 mL microcentrifuge tube Add 25 200 uL of prewarmed 65 C 70 C 10 mM Tris HCL pH 8 5 or prewarmed water to the column See Elution Parameters pages 6 7 to determine the suitable elution volume for your application Note Make certain that the elution buffer covers the entire membrane Continued on next page Purifying gDNA from Tissue Continued Purifying 12 DNA 13 Continued 14 15 Incubate at room temperature for 5 minutes Centrifuge the column at 12 000 x g for 2 minutes at room temperature The tube contains purified genomic DNA
11. the JetQuick Tissue DNA Miniprep Kit is described below The fixative used influences the yield and quality of the purified DNA Fixatives such as alcohols or formalin are easiest to handle For tissues fixed with paraffin a pre treatment of the sample is necessary see page 37 Fixatives that cause cross linking i e osmic acid are not recommended as it can be difficult to obtain amplifiable DNA from tissues fixed with these agents To obtain high quality genomic DNA follow the guidelines recommended on page 4 e Xylene or PBS e 96 100 6 ethanol e Sample for DNA isolation e Sterile DNase free microcentrifuge tubes e Water baths or heat blocks e Microcentrifuge capable of centrifuging gt 12 000 x g Components supplied with the JetQuick Tissue DNA Miniprep Kit e Buffers T1 T2 T3 and TX e Proteinase K lyophilized powder see Before Starting page5 e RNase A 20 mg mL e 10mM Tris HCl pH 8 5 e JetQuick Spin Columns e JetQuick Receiver Tubes Continued on next page Purifying gDNA from Paraffin Embedded Tissue Continued Note Xylene Extraction Formalin Alcohol Fixed Tissues Formalin Fixed Paraffin Embedded Tissue An RNase A digestion step is normally not necessary for fixed tissues Due to the age of the sample and prior treatments most of the RNA is already degraded It may not be necessary to remove the paraffin by xylene extraction before processing The paraffin
12. uL 3mL 10 mL Note Mix the sample immediately to prevent precipitation of nucleic acids due to high local alcohol concentrations Proceed immediately to Purifying DNA next page Continued on next page 19 Purifying gDNA from Blood and Body Fluids Using Vacuum Continued Purifying DNA 20 1 Attach vacuum manifold such as the EveryPrep Universal Vacuum Manifold see page 57 to a vacuum source Attach as many JetQuick Spin Columns as necessary to the female Luer inlets on the vacuum manifold Load the blood lysate from Step 6 previous page into the JetQuick Spin Columns Apply vacuum 200 to 650 mbar until all liquid is pulled through the column Turn off the vacuum source Add reconstituted Buffer KX page 5 to the column Reagent Miniprep Midiprep Maxiprep Buffer KX 500 uL 10 mL 10 mL Apply vacuum 200 to 650 mbar until all liquid has been pulled through the column Turn off vacuum source Add Buffer K2 prepared with ethanol page 5 to the column and repeat Step 5 Reagent Miniprep Midiprep Maxiprep Buffer K2 500 uL 10 mL 10 mL To remove last traces of ethanol turn on the vacuum again and pull air through the spin columns on the vacuum manifold until all residual ethanol is removed from the silica membrane Place the JetQuick Spin Column into a new sterile elution tube not supplied Spin Column Miniprep M
13. water bath or heat block at 58 C Miniprep or 70 C Midiprep and Maxiprep 1 Harvest cells into the appropriate tube see table below e For cells grown in a monolayer remove the growth medium from the culture plate and harvest cells by trypsinization or use a cell scraper according to established protocols Transfer cells into a harvesting tube see table below e For cells grown in suspension transfer cells into a harvesting tube Starting Miniprep Midiprep Maxiprep Materials Harvesting i5mp s50mL 50mL tube Centrifuge at 300 350 x g for 5 minutes to pellet cells Remove the growth medium completely without disturbing the cell pellet and resuspend the cells in PBS Reagent Miniprep Midiprep Maxiprep PBS 200 uL 3mL 10mL Continued on next page 23 Purifying gDNA from Mammalian Cells Continued Preparing Cell Lysate Continued 24 Add the reagents in the order listed in the table below and mix thoroughly by vortexing or inverting the tube Do not add Genomed Protease directly to Buffer K1 First mix the suspended cells with the enzyme and then add Buffer K1 Reagent Miniprep Midiprep Maxiprep Operation Genomed Protease 20 uL 300 uL 500 uL 20 mg mL Optional RNase A 20 mg mL 10 uL 100 uL 300 uL Mix sample vortex vortex vortex Buffer K1 200 uL 3mL 10 mL M
14. Continued Purifying DNA Continued Storing DNA 16 12 Add prewarmed 70 C 10 mM Tris HCl pH 8 5 or water to the column See Elution Parameters pages 6 7 to determine the suitable elution volume for your application Incubate at room temperature for the time specified in the 13 14 table below Elution Miniprep Midiprep Maxiprep 10 mM Tris HCI pH 8 5 25 200 uL 500 800 uL 1 4mL Incubation R 2 minutes 5 minutes 5 minutes Time Centrifuge the col umn at room temperature to elute the DNA Centrifugation Miniprep Midiprep Maxiprep Acceleration 10 000 xg 5 000 x g 5 000 x g Time 2minutes 2 minutes 2 minutes Optional To recover more DNA perform a second elution in another sterile tube Centrifuge the column as directed in Step 13 The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application To avoid repeated freezing and thawing of DNA Store the purified DNA at 4 C for immediate use or Aliquot the DNA and store at 20 C for long term storage Purifying gDNA from Blood and Body Fluids Using Vacuum Introduction The procedure for purifying genomic DNA from blood and any type of body fluid e g amniotic fluid saliva sperm lymph us
15. Duration 1 minute 3 minutes 3 minutes Discard the flow through Add reconstituted Buffer KX page 5 to the column Reagent Miniprep Midiprep Maxiprep Buffer KX 500 uL 10 mL 10 mL Centrifuge using the following conditions Centrifugation Miniprep Midiprep Maxiprep Acceleration 10 000 xg 5 000 x g 5 000 x g Duration 1 minute 2 minutes 2 minutes Continued on next page Purifying gDNA from Blood and Body Fluids Using Centrifugation Continued Purifying DNA 7 Discard the flow through and reassemble the spin column Continued Add Buffer K2 prepared with ethanol page 5 to the column Reagent Miniprep Midiprep Maxiprep Buffer K2 500 uL 10 mL 10 mL 9 Centrifuge using the conditions described in Step 6 10 Discard the flow through and re assemble the spin column using the same receiver tube Centrifuge again to clear the silica membrane of any residual liquid Discard the receiver tube Centrifugation Miniprep Midiprep Maxiprep Acceleration 12 000 xg 5 000 x g 5 000 x g Duration 1 minute 10 minutes 10 minutes 11 Place the JetQuick Spin Column into a new sterile elution tube not supplied Spin Column Miniprep Midiprep Maxiprep Assembly Fluti ntabe a Sai 50 mL 50 mL size Continued on next page 15 Purifying gDNA from Blood and Body Fluids Using Centrifugation
16. ES Genomed JetQuick Genomic DNA Purification Kits For purification of genomic DNA from blood mammalian cells tissue swabs fixed tissue buffy coat body fluids bacteria and yeast Catalog nos 440050 440250 441020 441050 442020 442050 450050 450250 Rev Date 12 A ug 2010 Manual Part no 70 15016 MAN 0001742 User Manual Contents Kit Contents and Stora gessneri inini iv Introduction 2i tere tun eo cete von a rauu b inr roa ra aro D qure T 1 System OVERVIEW c ciae red esthite iater NARVA tne es 1 Experimerital Outline iced etiem ines 3 Method er 4 General Information eese tette RE 4 Purifying gDNA from Blood and Body Fluids Using Centrifugation 8 Purifying gDNA from Blood and Body Fluids Using Vacuum 17 Purifying gDNA from Mammalian Cells sse 22 Purifying gDNA from Tissue esseeeeeeeneenen eene en nennen 28 Purifying gDNA from Swabs Purifying gDNA from Paraffin Embedded Tissue 36 Purifying gDNA from Bacteria sse eene 41 Purifying gDNA from Yeast cette teretes 47 Examples of Expected Results sse nee 52 Troubleshooting t ete intei e ees 54 Appen diN raea aee aaa ae eaa aaraa aaa aeiaai dai Eea diasakan 57 Accessory Products Technical SUpport nee ne etienne ete nine Purchaser Notification aco tete c Eve lee ee a e E ED RD d 59 Kit Conten
17. III EcoRI Lane 2 DNA isolated from bovine liver Lane 3 DNA isolated from bovine spleen Lane 4 DNA isolated from bovine pancreas Lane 5 DNA isolated from porcine kidney Lane 6 DNA isolated from porcine spleen Lane 7 DNA isolated from chicken liver 12345678 23 Kb 2 Kb 53 Troubleshooting Refer to the table below to troubleshoot problems that you may encounter when using the JetQuick genomic DNA Purification kits Introduction Problem Cause Solution Low DNA yield Incomplete lysis e Decrease the amount of starting material used e Add Genomed Protease or Proteinase K during lysis e Increase the digestion time or amount of Genomed Protease or Proteinase K used for lysis Poor quality of starting material Use fresh samples and process immediately after collection or freeze the samples at 80 C or in liquid nitrogen The yield and quality of DNA isolated is dependent on the type and age of the starting material Incorrect binding conditions Add 96 100 ethanol to the lysate prior to loading the samples on the spin column Mix the sample by vortexing Avoid overloading the column Ethanol not added to Buffers KX and K2 JetQuick Blood amp Cell Culture DNA Kits or Buffers TX and T3 JetQuick Tissue DNA Miniprep Kits Add 96 10096 ethanol to Wash Buffers KX and K2 or Buffers TX and T3 as indicated on the label 54 Continue
18. Prej pare lysate with Genomed Protease i CC ami C aj 6 can CV and RNase A Add 200 uL ethanol to the lysate Apply sample to a JetQuick Spin Column Wash the column with 500 uL Buffer KX Wash the column with 500 uL Buffer K2 Elute with Tris HCl pH 8 5 MidiPrep Prepare lysate with Genomed Protease and RNase A Add 3 mL ethanol B to the lysate y Apply sample to a JetQuick Spin Column Wash the column with 10 mL Buffer KX nd ba Wash the column l with 10 mL Buffer K2 w ba Elute with Tris HCI pH 8 5 The figure below illustrates the basic steps necessary to purify gDNA using centrifugation with the JetQuick Blood amp Cell Culture DNA Purification Kits MaxiPrep Prepare lysate with Genomed Protease and RNase A Add 10 mL ethanol a to the lysate Apply sample to a es JetQuick Spin Column wu Y Ld Wash the column with 10 mL Buffer KX wu v He Wash the column s With 10 mL Buffer K2 Ra Elute with Tris HCl pH 8 5 Continued on next page Purifying gDNA from Blood and Body Fluids Using Centrifugation Continued Sample Volumes 10 The table below lists the appropriate procedure and the relevant page reference based on the starting sample volume Starting Prep Size Protocol Page Volume no Up to 200 uL Miniprep Standard no 12 modification 300 uL 1 mL Miniprep Requires lysate 11
19. Preparing Bacterial Lysate next page Continued on next page 43 Purifying gDNA from Bacteria Continued Preparing Bacterial Lysate 44 Set one water bath or heat block at 56 C and another at 70 C Add 20 pL Proteinase K 20 mg mL to the to the sample from Step 3 Harvesting Bacteria from Biological Fluids page 42 or Harvesting Gram Negative Bacteria page 43 or Step 5 Harvesting Bacteria from Swabs page 42 or Harvesting Gram Positive Bacteria page 43 Mix thoroughly by inverting the tube several times Incubate at 56 C with occasional vortexing until mixture is clear indicating lysis is complete 1 to 2 hours Note Reduce incubation time to 30 minutes for Gram Positive Bacterial Cell Lysates To remove any particulate materials centrifuge the lysate at 10 000 x g for 3 minutes at 4 C Transfer supernatant to a new sterile microcentrifuge tube Optional Add 20 pL RNase A 20 mg mL to the lysate and mix well by brief vortexing Add 200 pL Buffer T2 and mix well by vortexing Incubate at 70 C for 10 minutes Cool the mixture at room temperature for 1 minute Add 200 pL 96 100 ethanol to the lysate Mix immediately and thoroughly by vortexing for 5 seconds Note When processing multiple samples you may prepare a Master Buffer Ethanol Mix by pre mixing 200 pL Buffer T2 and 200 uL 96 100 ethanol for each sample Proceed immediately to Purifying DNA next page Continued on next page P
20. RN 100 Products below are available from Invitrogen See www invitrogen com Phosphate Buffered Saline PBS 1X 500 mL 10010 023 EveryPrep Universal Vacuum Manifold 1 unit K211101 E Gel E Gel Agarose Gels are bufferless pre cast agarose gels Agarose Gels designed for fast convenient electrophoresis of DNA samples and DNA E Gel agarose gels are available in different agarose Ladders percentages and well formats for your convenience DNA ladders are available from Invitrogen for sizing DNA For more details on these products visit www invitrogen com 57 Technical Support World Wide Visit the website at www genomed dna com for Web e Technical resources including manuals SDSs FAQs e Complete technical support contact information e Access to the Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on www genomed dna com GENOMED GmbH Poststr 22 D 32584 L hne Germany Tel 49 0 5732 904700 Fax 49 0 5732 9047010 E mail techservice genomed dna com 58 Purchaser Notification Limited Warranty GENOMED a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have
21. ain that the elution buffer comes into contact with the entire membrane Continued on next page 45 Purifying gDNA from Bacteria Continued Purifying DNA Continued Storing DNA 46 14 15 Incubate at room temperature for 5 minutes 13 Centrifuge the column at 12 000 x g for 2 minutes at room temperature The tube contains purified genomic DNA Optional To recover more DNA perform a second elution step in another sterile 1 5 mL microcentrifuge tube Centrifuge the column at 12 000 x g for 2 minutes at room temperature The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application To avoid repeated freezing and thawing of DNA Store the purified DNA at 4 C for immediate use or Aliquot the DNA and store at 20 C for long term storage Purifying gDNA from Yeast Introduction The procedure for purifying gDNA from yeast using the JetQuick Tissue DNA Miniprep Kit is described below The protocol has been modified from the publication Boeke J D Garfinkel D J Styles C A and Fink G R 1985 Ty elements transpose through an RNA intermediate Cell 40 491 To obtain high quality genomic DNA follow the guidelines recommended on page 4 Materials e Buffer Y1 Needed e 96 100 ethanol e Sa
22. aliva sperm lymph mouse Trat tail swabs bacteria yeast and fixed tissue The isolated DNA is 20 50 kb in size and is suitable for PCR restriction enzyme digestion and Southern blotting JetQuick spin columns contain silica membranes that bind nucleic acids within columns under defined conditions Using this system purify DNA in the following manner 1 Lysenuclei and denature proteins e g nucleases histones in the presence of a cell lysis buffer 2 Digest denatured proteins into smaller fragments and strip genomic DNA of all bound proteins with protease 3 Remove residual RNA by digestion with RNase A prior to sample binding to the silica membrane 4 Bind DNA to the membrane in the column in the presence of chaotropic salts Remove impurities by thorough washing with Wash Buffers 5 Elute gDNA in 10 mM Tris HCl pH 8 5 Continued on next page System Overview Continued Advantages The advantages of using JetQuick Genomic DNA Purification Kits are e Rapid and efficient purification of high quality genomic DNA from a variety of samples such as mammalian cells and tissue blood samples any type of body fluid e g amniotic fluid saliva sperm lymph mouse tails swabs bacteria yeast and fixed tissue e Simple lysis of cells and tissues with Proteinase K or Genomed Protease without the need for any mechanical lysis e Minimal contamination from RNA e Reliable performan
23. am enzymatic reactions To remove Wash Buffers K2 or T3 from JetQuick Spin Column discard Wash Buffer flow through Centrifuge the JetQuick Spin Column at maximum speed for up to 15 minutes or incubate the spin columns for 10 minutes at 70 C in an incubator to evaporate residual ethanol Presence of salt in Use the correct order of Wash contamination applies to Purifying gDNA from Blood and Body Fluids Using Vacuum only purified DNA Buffers for washing Maintain a ratio of 1 1 1 for Sample Binding Buffer K1 or T2 Ethanol Low elution volume Incorrect vacuum Make sure the vacuum manifold or sample cross pressure is sealed tightly without leakage Maintain the vacuum pressure at 6 to 12 inches Hg 200 to 400 mbar or 150 to 300 mm Hg to obtain the best results 56 Appendix Accessory Products Additional The table below lists additional products available from Products Genomed or Invitrogen that may be used with the JetQuick genomic DNA Purification Kits Product Amount Cat no Products below are available from Genomed See www genomed dna com Buffer K1 200 mL K1 200 Buffer K2 150 mL K2 500 Buffer KX 216 mL KX 500 Buffer T1 200 mL T1 200 Buffer T2 200 mL T2 200 Buffer T3 150 mL T3 500 Buffer TX 216 mL TX 500 Proteinase K 40 mg GN PK 040 200 mg GN PK 200 Ribonuclease A 50 mg GN RN 50 100 mg GN
24. and re assemble the JetQuick Spin Column using the same 2 mL Receiver Tube Centrifuge the column again at 12 000 x g for 1 minute at room temperature to clear the silica membrane of any residual liquid Discard the receiver tube Place the JetQuick Spin Column into a sterile 1 5 mL microcentrifuge tube Add 25 200 uL of prewarmed 65 C 70 C 10 mM Tris HCL pH 8 5 or prewarmed water to the column Refer to pages 6 7 to determine elution volume Incubate at room temperature for 5 minutes Continued on next page Purifying gDNA from Yeast Continued Purifying 13 DNA Continued 14 15 Centrifuge the column at 12 000 x g for 2 minutes at room temperature The tube contains purified genomic DNA Optional To recover more DNA perform a second elution step in another sterile 1 5 mL microcentrifuge tube Centrifuge the column at 12 000 x g for 2 minutes at room temperature The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application Storing DNA To avoid repeated freezing and thawing of DNA Store the purified DNA at 4 C for immediate use or Aliquot the DNA and store at 20 C for long term storage 51 Examples of Expected Results Blood gDNA Genomic DNA from various samples was isolated using a
25. any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All GENOMED products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order GENOMED makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Conti
26. ation Incubate at room temperature for the time specified in the table below Elution Miniprep Midiprep Maxiprep 10 mM Tris HCI pH 8 5 25 200 uL 500 800 pL 1 4 mL Incubation y 2 minutes 5 minutes 5 minutes time Continued on next page 26 Purifying gDNA from Mammalian Cells Continued Purifying DNA 13 Centrifuge the column at room temperature to elute the Continued DNA Centrifugation Miniprep Midiprep Maxiprep Acceleration 10 000 x g 5 000 x g 5 000 x g Time 2minutes 2minutes 2 minutes 14 Optional To recover more DNA perform a second elution step in another sterile tube Centrifuge the column as directed in Step 13 The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application Storing DNA To avoid repeated freezing and thawing of DNA e Store the purified DNA at 4 C for immediate use or e Aliquot the DNA and store at 20 C for long term storage 27 Purifying gDNA from Tissue Introduction The procedure for purifying gDNA from mammalian tissues including mouse tail using JetQuick Tissue DNA Miniprep Kits is described below The protocol was adapted from the method published by Bowtell see Bowtell D L L 1987 Rapid is
27. bs 42 Gram negative cultures 43 Gram positive cultures 43 For the isolation of gDNA from eye nasal or pharyngeal swabs or from gram positive cultures additional buffers and enzymes are necessary see relevant protocols for more information e PBS and fungicide for Harvesting Bacteria from Swabs e Lysozyme Digestion Buffer for Harvesting Gram Positive Bacteria e 96 100 6 ethanol e Sample for DNA isolation e Sterile DNase free microcentrifuge tubes e Water baths or heat blocks e Microcentrifuge capable of centrifuging gt 12 000 x g Continued on next page 41 Purifying gDNA from Bacteria Continued Components supplied with the JetQuick Tissue DNA Miniprep Kit Harvesting Bacteria from Biological Fluids Harvesting Bacteria from Swabs 42 Buffers T1 T2 T3 and TX Proteinase K lyophilized powder see page 5 RNase A 20 mg mL 10 mM Tris HCl pH 8 5 JetQuick Spin Columns and JetQuick Receiver Tubes Harvest bacteria by centrifugation at 12 000 x g for 3 minutes Remove the clarified supernatant completely with a pipette Resuspend the cell pellet in 200 uL Buffer T1 Proceed immediately to Preparing Bacterial Lysate page 44 d Place sample in 2 mL PBS containing fungicide Incubate at room temperature for several hours Harvest bacteria by centrifugation at 12 000 x g for 3 minutes Remove the clarified supernatant completely with a pipette Resuspe
28. card the supernatant and resuspend in 2 mL buffer Y1 see Step 1 4 Centrifuge again at 3 000 5 000 x g for 5 10 minutes at 4 C Discard the supernatant Resuspend the cells in 1 mL Buffer Y1 5 Add 20 units of Zymolyase 100T MP Biomedicals 100 000 units g and incubate at 37 C for 20 to 30 minutes Note Monitor spheroplast formation by examining detergent sensitivity dilute a small sample of cells in 1 SDS Spheroplasting is sufficient when greater than 90 of the cells burst when examined under the microscope Continued on next page Purifying gDNA from Yeast Continued Preparing 6 Yeast Lysate Continued 7 10 11 12 13 14 15 Centrifuge the spheroplasts at 5 000 x g for 10 minutes at 4 C Discard the supernatant Resuspend the spheroplasts in 200 uL Buffer T1 in a 1 5 mL microcentrifuge tube Mix thoroughly by inverting the tube several times Add 20 pL Proteinase K 20 mg mL to the tube Mix thoroughly by inverting the tube several times Note When processing multiple samples you may prepare a master Digestion Buffer Mix by combining 200 uL Buffer T1 and 20 uL Proteinase K for each sample Incubate with occasional vortexing until lysis is complete 1 to 2 hours or overnight at 56 C To remove any particulate materials centrifuge the lysate at 10 000 x g for 3 minutes at 4 C Transfer supernatant to a new sterile microcentrifuge tube Optional Add 20 uL RNase A 20 mg mL t
29. ce of the purified DNA in PCR restriction enzyme digestion and Southern blotting System The table below lists the specifications for JetQuick Genomic Specifications DNA Purification Kits Kit Type Blood Tissue Kit Size Miniprep Midiprep Maxiprep Miniprep Starting Material 200 1 000 uL 1 5 mL 5 20 mL varies whole blood whole blood whole blood Binding Capacity 50 ug 150 pg 600 ug 50 ug Column Reservoir 800 uL 15 mL 15 mL 800 pL Capacity Elution Volume 25 200 pL 500 800 uL 1 4 mL 25 200 uL DNA Yield 85 9596 85 9596 85 9596 85 9596 DNA Size up to 50 kb up to 50 kb up to 50 kb up to 50 kb Experimental Outline Introduction The figure below illustrates the basic steps necessary to use JetQuick gDNA Purification Kits For a more detailed overview of blood gDNA purification refer to page 9 Purifying gDNA from Blood and Body Fluids Using Centrifugation or page 17 Purifying gDNA from Blood and Body Fluids Using Vacuum Prepare Lysate using Buffer Protease and RNase A Add Ethanol to the Lysate Apply sample to Spin Column Wash column Repeat Wash Step Elute DNA with 10 mM Tris HCl pH 8 5 x 7 9 GI Go mi 1 Methods General Information NAN ev O m e EN Nowe Follow the recommendations below to obtain the best results e Maintain a sterile environment when handling DNA to avoid any contamination from DNases and ensure that no DNases are introduced int
30. d on next page Troubleshooting Continued Problem Cause Solution Low DNA yield Incorrect elution Add 10 mM Tris HCI pH 8 5 conditions and perform incubation with buffer before centrifugation To recover more DNA perform a second elution step DNA is sheared or Avoid repeated freezing and degraded thawing of samples to prevent any DNA damage Maintain a sterile environment while working to avoid any contamination from DNases Dark colored eluate or discolored membrane mammalian tissue mouse tails or blood samples only Pigments from tissues or heme from blood bind to the silica matrix and co elute with DNA Be sure to add ethanol to the lysate prior to loading the lysate on the JetQuick Spin Columns The ethanol prevents the pigments from sticking on the silica matrix Perform centrifugation of the lysate at a higher speed and longer time prior to loading the lysate on to the column RNA contamination Silica membrane binds total nucleic acid present in the sample Perform RNase A digestion step during sample preparation Continued on next page 55 Troubleshooting Continued enzymatic reactions Problem Cause Solution Inhibition of Presence of ethanol in Traces of ethanol from the Wash downstream purified DNA Buffer K2 JetQuick Blood amp Cell Culture DNA Kits or T3 JetQuick Tissue DNA Miniprep Kits can inhibit downstre
31. e A 20 mg mL optional to the sample and mix very thoroughly by vortexing or inverting the tube Add 600 pL Buffer K1 and mix very thoroughly by vortexing or inverting the tube Incubate at 58 C for 10 minutes Add 600 uL of 96 100 ethanol Mix well by vortexing to obtain a homogenous solution Note Mix the sample immediately to prevent the precipitation of nucleic acids due to high local alcohol concentrations Proceed immediately to Purifying DNA next page Continued on next page 33 Purifying gDNA from Swabs Continued Purifying DNA 34 1 10 11 12 Assemble a JetQuick Spin Column with a 2 mL Receiver Tube Apply 700 uL of the sample from Step 9 previous page into the JetQuick Spin Column Centrifuge the sample for 1 minute at 10 000 x g Discard the flow through Note Repeat Steps 1 3 until all liquid from Step 9 has been processed over the JetQuick Spin Column Squeeze out residual liquid from the swab and discard the swab Add 500 pL Buffer KX with ethanol page 5 to the column Centrifuge the column at room temperature at 10 000 x g for 1 minute Discard the flow through and re assemble the JetQuick Spin Column Add 500 pL Buffer K2 with ethanol page 5 to the column Centrifuge the column at room temperature at 10 000 x g for 1 minute Discard the flow through and re assemble the JetQuick Spin Column using the same 2 mL Receiver Tube Centrifuge the column agai
32. g mL to the tube Mix thoroughly by inverting the tube several times Note When processing multiple samples you may prepare a master Digestion Buffer Mix by pre mixing 200 uL Buffer T1 and 20 pL Proteinase K for each sample Incubate at 55 C with occasional vortexing until mixture is clear indicating complete lysis 1 2 hours For mouse tails or larger tissue pieces incubate overnight To remove any particulate materials centrifuge the lysate at 10 000 x g for 5 minutes at 4 C Transfer clear supernatant to a new sterile microcentrifuge tube Optional Add 20 uL RNase A 20 mg mL to the lysate and mix well by brief vortexing Add 200 pL Buffer T2 and mix well by vortexing Incubate at 70 C for 10 minutes Cool the mixture at room temperature for 1 minute Add 200 pL 96 100 ethanol to the lysate Mix immediately and thoroughly by vortexing for 5 seconds Note When processing multiple samples you may prepare a Master Buffer Ethanol Mix by pre mixing 200 uL Buffer T2 and 200 pL 96 100 ethanol for each sample Proceed immediately to Purifying DNA next page Continued on next page 29 Purifying gDNA from Tissue Continued Purifying DNA 30 1 10 11 Assemble a JetQuick Spin Column with a 2 mL Receiver Tube Apply the sample from Step 10 previous page into the JetQuick Spin Column Note Processing too many cells may lead to a high DNA content in the sample so that DNA may already be partially
33. gh Add Buffer KX with ethanol page 5 to the column Reagent Miniprep Midiprep Maxiprep Buffer KX 500 uL 10 mL 10 mL Centrifuge using the following conditions Centrifugation Miniprep Midiprep Maxiprep Acceleration 10 000 x g 5 000 x g 5 000 x g Time 1 minute 2 minutes 2 minutes Discard the flow through and reassemble the spin column Continued on next page 25 Purifying gDNA from Mammalian Cells Continued Purifying DNA 8 Add Buffer K2 reconstituted with ethanol see page 5 to Continued the column Reagent Miniprep Midiprep Maxiprep Buffer K2 500 pL 10 mL 10 mL 9 Centrifuge using the conditions described in Step 6 10 Discard the flow through and re assemble the spin column using the same receiver tube Centrifuge again to clear the silica membrane of any residual liquid Discard the receiver tube Centrifugation Miniprep Midiprep Maxiprep Acceleration 12 000 x g 5 000 x g 5 000 x g Time 1 minute 10 minutes 10 minutes 11 Place the JetQuick Spin Column into a new sterile elution tube Spin Column Miniprep Midiprep Maxiprep Assembly Elution tube 1 5mL 50mL 50mL size 12 Add prewarmed 70 C 10 mM Tris HCl pH 8 5 or prewarmed water to the column See Elution Parameters pages 6 7 to determine the suitable elution volume for your applic
34. idiprep Maxiprep Assembly Elution tube 15 mL 50 mL 50 mL size Continued on next page Purifying gDNA from Blood and Body Fluids Using Vacuum Continued Purifying DNA Continued Storing DNA 9 10 Lt Add prewarmed 70 C 10 mM Tris HCl pH 8 5 or water to the column See Elution Parameters pages 6 7 to determine the suitable elution volume for your application Incubate at room temperature for the time specified in the table below Elution Miniprep Midiprep Maxiprep 10 mM Tris HCl pH 8 5 25 200 pL 500 800 uL 1 4 mL Incubation 2 minutes 5 minutes 5 minutes Time Centrifuge the col umn at room temperature to elute the DNA Centrifugation Miniprep Midiprep Maxiprep Acceleration 10 000 xg 5 000 x g 5 000 x g Time 2minutes 2 minutes 2 minutes The tube contains purified genomic DNA Optional To recover more DNA perform a second elution in another sterile tube Centrifuge the column as directed in Step 10 The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application To avoid repeated freezing and thawing of DNA Store the purified DNA at 4 C for immediate use or Aliquot the DNA and store at 20 C for long te
35. ing JetQuick Blood amp Cell Culture DNA Kits with a vacuum manifold is described below To obtain high quality genomic DNA follow the guidelines recommended on page 4 Experimental The figure below illustrates the basic steps necessary to Outline purify gDNA using vacuum with the JetQuick Blood amp Cell Culture DNA Kits Prepare lysate using Genomed Protease and RNase A Add ethanol to the lysate Apply sample to JetQuick Spin Column attached to vacuum manifold i Wash column with Buffer KX Vacuum J Wash column with Buffer K2 Vacuum I Elute DNA with 10 mM Tris HCl pH 8 5 v Continued on next page 17 Purifying gDNA from Blood and Body Fluids Using Vacuum Continued Materials Needed Preparing Lysate 18 96 100 ethanol Sample for DNA isolation see page 10 Sterile DNase free microcentrifuge tubes Water baths or heat blocks Microcentrifuge capable of centrifuging gt 12 000 x g Vacuum manifold see page 57 Components supplied with the JetQuick Blood amp Cell Culture DNA Kits Buffers K1 K2 and KX Genomed Protease lyophilized powder see page 5 RNase A 20 mg mL 10 mM Tris HCl pH 8 5 JetQuick Spin Columns JetQuick Receiver Tubes Prepare lysate from up to 200 uL Mini 1 to 5 mL Midi or 6 to 10 mL Maxi blood or body fluid samples See page 10 for detailed starting sample volume information 1 Set a water bath or heat block at 58 C Mini or
36. its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 or outlicensing lifetech com 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Triton is a trademark of Union Carbide Corporation Corporate Headquarters Genomed GmbH Poststr 22 D 32584 L hne Germany Tel 49 0 5732 904700 Fax 49 0 5732 9047010 techservice
37. ix sample vortex vortex vortex Incubate the sample for 10 minutes to degrade protein Incubation Miniprep Midiprep Maxiprep Temperature 58 C 70 C 70 C Note The optimal incubation temperature for Genomed Protease is 58 C However to reach the catalytic temperature range as quickly as possible incubate Midiprep and Maxiprep samples at 70 C If incubation at 58 C is desired extend the incubation time to 20 minutes Add ethanol and mix well by vortexing Reagent Miniprep Midiprep Maxiprep 96 100 Ethanol 200 uL 3mL 10 mL Note Mix the sample immediately to prevent precipitation of nucleic acids due to high local alcohol concentrations Proceed immediately to Purifying DNA next page Continued on next page Purifying gDNA from Mammalian Cells Continued Purifying DNA 1 Assemble a JetQuick Spin Column with an appropriate receiver tube Spin Column Miniprep Midiprep Maxiprep Assembly Receiver Tibe 2mL 50mL 50mL Note For the Midiprep and Maxiprep loosely attach the cap to the tube to allow ventilation during the centrifugation Apply the sample from Step 7 previous page into the JetQuick Spin Column Centrifuge samples using the following conditions Centrifugation Miniprep Midiprep Maxiprep Acceleration 10 000 x g 2 000 x g 2 000 x g Time 1 minute 3 minutes 3 minutes Discard the flow throu
38. lysate from up to 200 uL Mini 1 to 5 mL Midi Lysate and 6 to 10 mL Maxi body fluid or blood samples nucleated or nonnucleated as described below See page 10 for alternate starting volumes that require lysate concentration 1 Seta water bath or heat block at 58 C for Miniprep or 70 C for Midiprep and Maxiprep 2 Harvest cells by adding fresh or frozen blood serum plasma or buffy coat to a clean sterile tube The table below indicates starting sample volumes Starting Miniprep Midiprep Maxiprep Materials Starting Sample 200 pL 3 mL 10 mL Volume Harvesting Tube 1 5 mL 50 mL 50 mL Size Continued on next page 12 Purifying gDNA from Blood and Body Fluids Using Centrifugation Continued Preparing Lysate Continued Add the reagents in the order listed in the table below and mix thoroughly by vortexing or inverting the tube Do not add Genomed Protease directly to Buffer K1 First mix the suspended cells with the enzyme and then add Buffer K1 Reagent Miniprep Midiprep Maxiprep Operation Genomed Protease 20 uL 300 uL 500 uL RNase A optional 10 uL 100 uL 300 uL Mix sample vortex vortex vortex Buffer K1 200 uL 3mL 10 mL Mix sample vortex vortex vortex Incubate the sampl e for 10 minutes at the temperature indicated in the table below to degrade protein Incubation Miniprep Midiprep Maxiprep Temperature
39. mL Buffer K2 10 mL 425mL 68 5mL 171mL 685mL 171 mL Buffer KX 12 mL 61 mL 95 mL 228 mL 99 mL 243 mL 10 mM Tris 22 mL 110mL 35mL 88mL 44 mL 110 mL HCL pH 8 5 RNase A 650 pL 3mL 3 mL 2x3mL 3x3mL 5x32mL 20 mg mL Genomed 22mg 5x22mg 5x26mg 320mg 220mg 560 mg Protease lyophilized powder JetQuick 50 each 250each 20each 50 each 20 each 50 each Spin Columns JetQuick 50 each 250 each none none none none Receiver Tubes 2 0 mL Continued on next page Kit Contents and Storage Continued JetQuick Tissue DNA Miniprep Kits vi The components supplied in the JetQuick Tissue DNA Miniprep Kits are listed below Store all components at room temperature Reagents 50 preps 250 preps 450 050 450 250 Buffer T1 11 mL 55mL Buffer T2 11 mL 55mL Buffer T3 10 mL 42mL Buffer TX 12 mL 60 mL 10 mM Tris HCl pH 8 5 22mL 110 mL RNase A 20 mg mL 1 5 mL 2x3mL Proteinase K lyophilized powder 26 mg 5 x 26 mg JetQuick Spin Columns 50 each 250 each JetQuick Receiver Tubes 2 0 mL 50 each 250 each Introduction System Overview Introduction How it Works JetQuick Genomic DNA Purification Kits allow rapid and efficient purification of genomic DNA gDNA The kits are designed to efficiently isolate genomic DNA from a variety of samples including blood mammalian cells and tissues body fluid e g amniotic fluid s
40. melts during the 56 C incubation and does not affect the JetQuick gDNA purification procedure However xylene extraction is necessary for some types of paraffin embedded samples You may try omitting the xylene extraction protocol since it makes the isolation procedure much simpler Wash formalin alcohol fixed tissue up to 30 mg tissue twice with PBS and proceed immediately to Preparing Fixed Tissue Lysate page 38 N Set a water bath or heat block at 37 C Place a small section of FFPE tissue no more than 30 mg in a sterile microcentrifuge tube Add 1 2 mL xylene to the sample and vortex vigorously for a few seconds Note Use appropriate precautions while using xylene and dispose of xylene in compliance with established institutional guidelines CitriSolv Clearing Agent Fisher catalog no 22 143 975 is a biodegradable alternative to xylene for paraffin extraction Centrifuge at 12 000 x g for 5 minutes at room temperature Carefully remove the supernatant without disturbing the pellet Continued on next page 37 Purifying gDNA from Paraffin Embedded Tissue Continued Formalin Fixed Paraffin Embedded Tissue Continued Before Starting Preparing Fixed Tissue Lysate 38 Add 1 2 mL 96 100 ethanol and vortex gently to remove residual xylene from the tissue pellet Centrifuge at 12 000 x g for 5 minutes at room temperature Carefully remove the supernatant Repeat ethanol ext
41. mple for DNA isolation e Sterile DNase free microcentrifuge tubes e Water baths or heat blocks e Microcentrifuge capable of centrifuging gt 12 000 x g Components supplied with the JetQuick Tissue DNA Miniprep Kit e Buffers T1 T2 T3 and TX e Proteinase K lyophilized powder see page 5 e RNase A 20 mg mL e 10 mM Tris HCl pH 8 5 e JetQuick Spin Columns e JetQuick Receiver Tubes Continued on next page 47 Purifying gDNA from Yeast Continued Zymolyase Preparing Yeast Lysate 48 Preparing the cell lysate includes an incubation step with Zymolyase see Step 5 below Zymolyase digests cell walls of yeast cells enzymatically during incubation An equivalent enzyme to Zymolyase is Lyticase Sigma Cat no L2524 Use concentrated Zymolyase but dilute Lyticase from the stock solution in distilled water to a final concentration of 1 000 U mL Perform Lyticase incubations for at least 30 minutes at 30 C Store stock solutions of both enzymes in aliquots at 20 C and use only once Check Buffer T1 for precipitates If present warm the solution briefly at 37 C to dissolve the precipitate Prepare lysate from yeast as described below 1 Prepare Buffer Y1 0 9 M sorbitol 0 1 M EDTA pH 7 5 14 mM f mercaptoethanol 2 Seta water bath or heat block at 56 C 3 Grow the Saccharomyces culture to saturation in YPD or YEPD Harvest by centrifugation at 3 000 5 000 x g for 5 to 10 minutes at 4 C Dis
42. mple is minimal e g mouse tail and RNA contamination does not interfere with any downstream applications of the purified DNA you may omit the RNase A digestion step during sample preparation Centrifugation Centrifuge Miniprep samples in a microcentrifuge e Centrifuge Midiprep and Maxiprep samples in a centrifuge with a swing out rotor Elution Elution Buffer Parameters gDNA is eluted using 10 mM Tris HCl pH 8 5 supplied with the kit Alternatively sterile water or TE can be used Elution Buffer Volume For increased DNA yield use a higher volume of elution buffer For increased DNA concentration use a lower volume of elution buffer Continued on next page General Information Continued Number of The absolute DNA yield can be increased by performing a Elutions second elution step Use the same volume of buffer for both elution steps To prevent dilution of the gDNA sample and to avoid contact of the spin column with the eluate perform the two elution steps using different tubes Prep Size Volume of DNA in DNA in First Elution 1 eluate 2 eluate Miniprep 50 uL 80 20 100 pL 90 10 Midiprep 0 3 mL 41 59 0 5 mL 60 40 0 8 mL 75 25 1 0 mL 83 17 Maxiprep 1 mL 80 20 2mL 87 13 3 mL 92 8 4mL 93 7 Purifying gDNA from Blood and Body Fluids Using Centrifugation Introduction Materials Needed The JetQuick Blood
43. n at 12 000 x g for 1 minute at room temperature to clear the silica membrane of any residual liquid Discard the receiver tube Place the JetQuick Spin Column into a sterile 1 5 mL microcentrifuge tube Add 25 200 uL of prewarmed 65 C 70 C 10 mM Tris HCI pH 8 5 or prewarmed water to the column See Elution Parameters pages 6 7 to determine the suitable elution volume for your application Incubate at room temperature for 2 minutes Continued on next page Purifying gDNA from Swabs Continued Purifying 13 DNA Continued 14 15 Centrifuge the column at 10 000 x g for 2 minutes at room temperature The tube contains purified genomic DNA Optional To recover more DNA perform a second elution step in another sterile 1 5 mL microcentrifuge tube Centrifuge the column at 10 000 x g for 2 minutes at room temperature The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application Storing DNA To avoid repeated freezing and thawing of DNA Store the purified DNA at 4 C for immediate use or Aliquot the DNA and store at 20 C for long term storage 35 Purifying gDNA from Paraffin Embedded Tissue Introduction Materials Needed 36 The procedure for purifying gDNA from fixed tissues using
44. nd the cell pellet in 200 uL Buffer T1 Proceed immediately to Preparing Bacterial Lysate page 44 Continued on next page Purifying gDNA from Bacteria Continued Harvesting Gram Negative Bacteria Harvesting Gram Positive Bacteria Harvest bacteria e For suspension cultures centrifuge at 12 000 x g for 3 minutes Remove the clarified supernatant completely with a pipette e For plate cultures remove bacterial cells from culture plate with an inoculation loop Suspend the bacterial cells in 200 pL Buffer T1 with vigorous stirring or vortexing Proceed immediately to Preparing Bacterial Lysate next page Gram positive bacteria require a pre incubation with specific enzymes such as Lysozyme or Lysostaphin Staphylococcus specific to disrupt the rigid multilayered murein cell wall For these species prepare the cell lysate as described below 1 2 Set a water bath or heat block at 37 C Prepare Lysozyme Digestion Buffer 20 mM Tris HCl pH 8 0 2mM EDTA 1 2 Triton X 100 To 200 pL Lysozyme Digestion Buffer sample add fresh Lysozyme or Lysostaphin to obtain a final Lysozyme concentration of 20 mg mL or Lysostaphin concentration of 200 pg mL Pellet up to 2 x 10 Gram positive cells by centrifugation at 12 000 x g for 3 minutes Resuspend the cell pellet in 200 pL Lysozyme Digestion Buffer with Lysozyme or Lysostaphin from Step 2 Incubate at 37 C for 30 minutes Proceed immediately to
45. nued on next page 59 Purchaser Notification Continued Limited Use Label License No 5 Invitrogen Technology 60 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or
46. o the lysate and mix well by brief vortexing Add 200 pL Buffer T2 and mix well by vortexing Incubate at 70 C for 10 minutes Then cool the mixture at room temperature for 1 minute Add 200 pL 96 100 ethanol to the lysate Mix immediately and thoroughly by vortexing for 5 seconds Note When processing multiple samples you may prepare a Master Buffer Ethanol Mix by pre mixing 200 pL Buffer T2 and 200 uL 96 100 ethanol for each sample Proceed immediately to Purifying DNA next page Continued on next page 49 Purifying gDNA from Yeast Continued Purifying DNA 50 1 10 11 12 Assemble a JetQuick Spin Column with a 2 mL Receiver Tube Apply the sample from Step 14 previous page into the JetQuick Spin Column Note Processing too many cells may lead to a high DNA content in the sample so that DNA may already be partially precipitated If any precipitated matter is present co transfer it into the spin column Centrifuge the sample for 1 minute at 10 000 x g Discard the flow through and re assemble the spin column into the 2 mL Receiver Tube Add 500 pL Buffer TX with ethanol see page 5 to the column Centrifuge the column at 10 000 x g for 1 minute at room temperature Discard the flow through and re assemble the spin column Add 500 pL Buffer T3 with ethanol page 5 to the column Centrifuge the column at 10 000 x g for 1 minute at room temperature Discard the flow through
47. o the sterile solutions of the kit e Make sure all equipment that comes in contact with DNA is sterile including pipette tips and microcentrifuge tubes e Do not vortex the samples for more than 5 10 seconds at each vortexing step to avoid extensive shearing of DNA e To minimize DNA degradation perform lysate preparation steps quickly and avoid repeated freezing and thawing of DNA samples e Perform all centrifugation steps at room temperature e Review Elution Parameters on pages 6 7 to determine the suitable elution volume for your requirements e Perform a 5 minute incubation step with 10 mM Tris HCl pH 8 5 e If you are using water for elution use sterile water pH 7 0 8 5 Continued on next page General Information Continued Before Starting Safety Information Reconstitute Buffers KX and K2 JetQuick Blood amp Cell Culture DNA Kits or TX and T3 JetQuick Tissue DNA Miniprep Kits with absolute ethanol as directed on the labels of the bottles Mix well Mark on each label that ethanol is added Store the wash buffers with ethanol at room temperature Resuspend Genomed Protease JetQuick Blood amp Cell Culture DNA Kits or Proteinase K JetQuick Tissue DNA Miniprep Kits in double distilled or Milli Q grade water to a final concentration of 20 mg mL Store the reconstituted enzymes in single use aliquots at 20 C Avoid repeated freezing and thawing Handle all blood and tissue samples in
48. olation of eukaryotic DNA Anal Biochem 162 463 To obtain high quality genomic DNA follow the guidelines recommended on page 4 Materials e 96 100 ethanol Needed e Sample for DNA isolation see below for recommended starting amount e Sterile DNase free microcentrifuge tubes e Water baths or heat blocks e Microcentrifuge capable of centrifuging gt 12 000 x g Components supplied with the JetQuick Tissue DNA Miniprep Kit e Buffers T1 T2 T3 and TX e Proteinase K lyophilized powder see page 5 e RNase A 20 mg mL e 10mM Tris HCl pH 8 5 e JetQuick Spin Columns e JetQuick Receiver Tubes Starting The table below lists the amount of starting material Material recommended based on tissue type Tissue Amount Brain Lung Heart Kidney 25 30 mg Liver Spleen 10 20 mg Mouse Tail 0 8 1 2 cm Continued on next page 28 Purifying gDNA from Tissue Continued Preparing Tissue Lysate Check Buffer T1 for precipitates If present warm the solution briefly at 37 C to dissolve the precipitate 1 10 11 Set one water bath or heat block at 55 C and another at 70 C Mince the tissue sample with a scalpel or freeze the tissue in liquid nitrogen and grind it into a fine powder with a mortar and pestle Add 200 pL Buffer T1 to the minced tissue sample in a 1 5 mL microcentrifuge tube Mix thoroughly by inverting the tube several times Add 20 pL Proteinase K 20 m
49. raction Steps 5 6 once Incubate the tubes with the lid open at 37 C for 10 to 15 minutes to evaporate residual ethanol Proceed immediately to Preparing Fixed Tissue Lysate below Check Buffer T1 for precipitates If present warm the solution briefly at 37 C to dissolve the precipitate Following fixative removal from the tissue sample prepare the lysate according to the following procedure 1 Set one water bath or heat block at 56 C and another at 70 C Add 200 pL Buffer T1 to the tissue sample Mix thoroughly by inverting the tube several times Add 20 uL Proteinase K 20 mg mL Mix well by inverting the tube several times Note When processing multiple samples you may prepare a master Digestion Buffer Mix by pre mixing 200 uL Buffer T1 and 20 uL Proteinase K for each sample Incubate at 56 C for 1 hour to overnight until mixture is clear indicating complete tissue digestion Mix the sample several times during the incubation for efficient digestion Continued on next page Purifying gDNA from Paraffin Embedded Tissue Continued Preparing Fixed Tissue Lysate Continued Purifying DNA Centrifuge the lysate at 10 000 x g for 3 minutes at 4 C to remove any particulate materials Transfer lysate to a new sterile microcentrifuge tube Add 200 pL Buffer T2 to the cleared lysate and mix well by vortexing Incubate at 70 C for 10 minutes Cool the mixture at room temperature for 1 minu
50. rm storage 21 Purifying gDNA from Mammalian Cells Introduction The procedure for purifying genomic DNA from mammalian cells using JetQuick Blood amp Cell Culture DNA Mini Midi and Maxiprep Kits is described below To obtain high quality genomic DNA follow the guidelines recommended on page 4 Materials e Needed n 96 100 ethanol Sample for DNA isolation see next page Phosphate Buffered Saline PBS page 57 Sterile DNase free microcentrifuge tubes Miniprep only Sterile DNase free 50 mL tubes Midiprep and Maxiprep Centrifuge with swing out rotor Midiprep and Maxiprep Microcentrifuge capable of centrifuging at 212 000 x g Water baths or heat blocks Components supplied with the JetQuick Blood amp Cell Culture DNA Mini Midi and Maxiprep Kits Buffers K1 K2 and KX Genomed Protease lyophilized powder see Before Starting page 5 RNase A 20 mg mL 10 mM Tris HCl pH 8 5 JetQuick Spin Columns JetQuick Receiver Tubes Miniprep Kits only 22 Continued on next page Purifying gDNA from Mammalian Cells Continued Starting Material Preparing Cell Lysate The table below lists the amount of starting material recommended for each purification kit Prep Size Number of cells Miniprep 5 x 10 to 1 x 10 Midiprep 7 5 x 107 to 1 x 108 Maxiprep 2 5 x 105 to 5 x 108 Prepare lysate from mammalian cells as described below Set a
51. tQuick Spin Columns e JetQuick Receiver Tubes The swab protocol utilizes an increased volume of Buffer K1 Therefore the standard 50 prep kit contains sufficient Buffer K1 to process 18 swab samples and the 250 prep JetQuick Blood amp Cell Culture DNA Miniprep Kit contains sufficient Buffer K1 to process 90 samples To utilize all other components included in the JetQuick Blood amp Cell Culture DNA Miniprep Kits purchase additional Buffer K1 separately see page 57 Note Continued on next page 32 Purifying gDNA from Swabs Continued Human Buccal Swab Lysate 1 10 Collect the buccal swab with a suitable tool such as a T swab Kit Isohelix Dacron swab Fitzco C E P Omni Swab Whatman or Cotton swab Puritan Hardwood Products according to standard collection procedures Nasal pharyngeal or vaginal swabs can be collected in a similar way Set a water bath or heat block at 58 C Place the buccal swab into a capped 2 mL microcentrifuge tube not provided with the kit e For C E P Omni swabs press the stem end toward the swab to eject it into the microcentrifuge tube e For swabs from other suppliers snap or cut the swab at the break point The swab should fit entirely inside the tube so that the cap may close Add 600 uL of PBS Tris Buffered Saline TBS or equivalent standard saline buffer Mix by vortexing Add 20 pL Genomed Protease 20 mg mL and 10 pL RNas
52. te Add 200 pL 96 100 ethanol to the lysate Mix immediately and thoroughly by vortexing for 5 seconds Note When processing multiple samples you may prepare a Master Buffer Ethanol Mix by pre mixing 200 pL Buffer T2 and 200 uL 96 100 ethanol for each sample Proceed immediately to Purifying DNA below Assemble a JetQuick Spin Column with a 2 mL Receiver Tube Apply the sample from Step 7 above into the JetQuick Spin Column Note Processing too many cells may lead to a high DNA content in the sample so that DNA may already be partially precipitated If any precipitated matter is present co transfer it into the spin column Centrifuge the sample for 1 minute at 10 000 x g Discard the flow through and re assemble the spin column into the 2 mL Receiver Tube Add 500 pL Buffer TX with ethanol see page 5 to the JetQuick Spin Column Centrifuge the JetQuick Spin Column at room temperature at 10 000 x g for 1 minute Discard the flow through and re assemble the JetQuick Spin Column Continued on next page 39 Purifying gDNA from Paraffin Embedded Tissue Continued Purifying DNA 7 Continued 10 11 12 13 Add 500 uL Buffer T3 with ethanol page 5 to the JetQuick Spin Column Centrifuge the JetQuick Spin Column at room temperature at 10 000 x g for 1 minute Discard the flow through and re assemble the spin column using the same receiver tube Centrifuge the column again at 12 000
53. ts and Storage Types of Kits This manual is supplied with the following products Product Quantity Cat no JetQuick Blood amp Cell Culture DNA 50 preps 440 050 Miniprep Kit 250 preps 440 250 JetQuick Blood amp Cell Culture DNA 20 preps 441 020 Midiprep Kit 50 preps 441 050 JetQuick Blood amp Cell Culture DNA 20 preps 442 020 Maxiprep Kit 50 preps 442 050 JetQuick Tissue DNA Miniprep Kit 50 preps 450 050 250 preps 450 250 Intended Use For research use only Not intended for any human or animal diagnostic or therapeutic uses Shipping and Each kit is shipped at room temperature Storage Upon receipt store all components at room temperature Note Genomed Protease Proteinase K and RNase A are stable when stored at room temperature However for optimal enzymatic performance store the enzymes at 4 C upon arrival For long term storage store Genomed Protease Proteinase K and RNase A at 20 C Continued on next page Kit Contents and Storage Continued JetQuick The components supplied in the JetQuick Blood amp Cell Blood amp Cell Culture DNA Kits are listed below Culture DNA Store all components at room temperature Kits Item Miniprep Midiprep Maxiprep 50 preps 250 preps 20 preps 50 preps 20 preps 50 preps 440 050 440 250 441 020 441050 442 020 442 050 Buffer K1 11 mL 55 mL 66 mL 165 mL 220 mL 550
54. urifying gDNA from Bacteria Continued Purifying DNA 1 10 11 Assemble a JetQuick Spin Column with a 2 mL Receiver Tube Apply the sample from Step 8 previous page into the JetQuick Spin Column Note Processing too many cells may lead to a high DNA content in the sample so that DNA may already be partially precipitated If any precipitated matter is present co transfer it into the spin column Centrifuge the sample for 1 minute at 10 000 x g Discard the flow through and re assemble the JetQuick Spin Column into the Receiver Tube Add 500 pL Buffer TX with ethanol see page 5 to the column Centrifuge the column at room temperature at 10 000 x 2 for 1 minute Discard the flow through and re assemble the JetQuick Spin Column Add 500 pL Buffer T3 with ethanol page 5 to the column Centrifuge the column at room temperature at 10 000 x g for 1 minute Discard the flow through and re assemble the JetQuick Spin Column using the same 2 mL Receiver Tube Centrifuge the column again at 12 000 x g for 1 minute at room temperature to clear the silica membrane of any residual liquid Discard the 2 mL Receiver Tube Place the JetQuick Spin Column into a sterile 1 5 mL microcentrifuge tube Add 25 200 uL of prewarmed 65 C 70 C 10 mM Tris HCL pH 8 5 or prewarmed water to the column See Elution Parameters pages 6 7 to determine the suitable elution volume for your application Note Make cert
55. x g for 1 minute at room temperature to clear the silica membrane of any residual liquid Discard the 2 mL Receiver Tube Place the JetQuick Spin Column into a sterile 1 5 mL microcentrifuge tube Add 25 100 uL of prewarmed 65 C 70 C 10 mM Tris HCL pH 8 5 or prewarmed water to the column See Elution Parameters pages 6 7 to determine the suitable elution volume for your application Note Make certain that the elution buffer covers the entire membrane Incubate at room temperature for 5 minutes Centrifuge the column at 12 000 x g for 2 minutes at room temperature The tube contains purified DNA Remove and discard the column Combine the contents of the elution tubes only if overall yield is of greater importance than maintaining the higher concentration obtained in the first eluate depending on your application Storing DNA To avoid repeated freezing and thawing of DNA Store the purified DNA at 4 C for immediate use or Aliquot the DNA and store at 20 C for long term storage 40 Purifying gDNA from Bacteria Introduction Materials Needed The procedure for purifying gDNA from various sources of bacterial species using the JetQuick Tissue DNA Miniprep Kit is described below Bacterial gDNA may be purified from biological fluids from eye nasal or pharyngeal swabs or from bacterial cultures see table below Source Page no Biological Fluids 42 Eye Nasal Pharyngeal Swa

CST Genomic DNA Purification Kits -- Tissues

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