ISOLATE II Genomic DNA Kit
Contents
1. e Preheat Elution Buffer G to 70 C A Bacterial cultures Collect bacteria using an inoculation loop Pellet bacteria by centrifugation for 5 min at 13 000 x g and remove the supernatant Proceed with step 2 pre lysis of the standard protocol see section 8 1 16 Product Manual www bioline com isolate B Clinical specimens nasal pharyngeal eye or other swabs Collect swab samples and place in 2ml PBS supplemented with an appropriate fungicide Incubate for 2 3 hours at room temperature Pellet bacterial cells by centrifugation for 5 min at 13 000 x g Proceed with step 2 pre lysis of the standard protocol see section 8 1 C Biological fluids Pellet bacteria by centrifugation for 5 min at 13 000 x g and remove the supernatant Proceed with step 2 pre lysis of the standard protocol see section 8 1 9 13 DETECTION OF MYCOBACTERIUM TUBERCULOSIS OR LEGIONELLA PNEUMOPHILA IN RESPIRATORY SAMPLES This protocol is suited for the detection of genomic DNA from M tuberculosis or L pneumophila in respiratory samples sputum or bronchoalveolar lavage Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 e Set an incubator or water bath to 56 C e Preheat Elution Buffer G to 70 C e Prepare N acetylcysteine NaOH 2g NaOH 1 45g sodium citrate 0 5g N acetylcysteine Add water to 100ml 1 Sample preparation Add 200 500uI sputum or bronchoalveolar lavage to an2. slightly alkaline pH 8 5 For high yields from large amounts of material we recommend elution with 200ul Elution Buffer G and incubation of the closed columns in an incubator at 70 C for 5 min before centrifugation RECOMMENDED SOLUTION Incomplete cell lysis Sample must be vortexed vigorously immediately after addition of Lysis Buffer GL Proteinase K solution Decreased Proteinase K activity store dissolved Proteinase K at 20 C for 6 months Reagents not applied correctly Prepare buffers and Proteinase K solution according to instructions section 7 1 Make sure ethanol is added to lysates before loading on columns RNA in sample eji ele cl zin Mero BU tS POSSIBLE CAUSE To remove RNA add 20yl RNase A solution 20mg ml not included before addition of lysis buffer RECOMMENDED SOLUTION Too much sample material Do not use more sample material than recommended in protocol If insoluble material remains in the lysate spin down the debris and transfer the clear supernatant to a new tube before proceeding with addition of Lysis Buffer G3 and ethanol ag Genomic DNA Kit Incomplete lysis Sample must be vortexed vigorously immediately after addition of Lysis Buffer GL Proteinase K solution Decreased Proteinase K activity store dissolved Proteinase K at 20 C for up to 6 months Reagents not applied correctly POSSIBLE CAUSE Prepare buffers and Proteinase K soluti
3. solution from buccal swabs Alternative Place an ISOLATE II Filter not provided into a Collection Tube Cut off the shaft of the buccal swab Transfer the buccal swab tip and any associated fluid onto the ISOLATE ll Filter Centrifuge for 1 min at 11 000 x g Discard ISOLATE II Filter and swab Or Alternative II Transfer as much of the lysate solution as possible into a 1 5ml microcentrifuge tube not provided Discard swab and continue with the recovered solution Lyse sample Add one volume of Lysis Buffer G3 400 or 600 ul this is dependent on the swab type and volume of PBS buffer used and vortex vigorously Incubate the samples at 70 C for 10 min Note Depending on the number of preparations additional Lysis Buffer G3 may be required Adjust DNA binding conditions Add one volume 96 100 ethanol 400 or 600yl depending on swab type to each sample and mix by vortexing Bind DNA Transfer 600yl of the samples into individual ISOLATE Il Genomic DNA Spin Columns Centrifuge at 11 000 x g for 1 min If the samples have not transferred through the column matrix completely repeat centrifugation Discard flow through Place columns back into Collection Tubes and repeat centrifugation with any remaining lysate When the entire lysate has been applied proceed to step 6 wash silica membrane of the standard protocol section 8 1 9 17 GENOMIC DNA FROM INSECTS Before you start Make sure Lysis Buffer G3 Wash Buffer GW2 and
4. that lyticase or zymolyase not supplied is available e Set an incubator or water bath to 30 C and 56 C e Preheat Elution Buffer G to 70 C occasionally To remove residual bone hair etc centrifuge for 5 min at 11 000 x g Transfer 200ul supernatant to a new centrifuge tube 3 Lyse sample Vortex sample briefly and add 200ul Lysis Buffer G3 Vortex vigorously i Sample preparati n Centrifuge 3ml YPD yeast culture OD lt 10 for 10 min at 5 000 x g Remove 4 Adjust DNA binding conditions supernatant 0 H ade Mol ethanol 96 1002 tothe sample Vortex vigorously Wash once with 1ml 10mM EDTA pH 8 and centrifuge for 10 min at 5 000 x g Proceed with step 5 of the standard protocol see section 8 1 2 Pre lysis Resuspend the pellet in 600Jj l sorbitol buffer and add 50U lyticase or zymolase 9 2 BACTERIA Incubate at 30 C for 30 min This step degrades the yeast cell wall creating Before you start spheroplasts Spheroplast formation may be checked microscopically e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see Note Concentration of lyticase or zymolyase can be increased up to 200U if spheroplasts are section 7 1 not found e For hard to lyse bacteria make up the following lysis buffer 20mM Tris HCl 2mM Centrifuge for 10 min at 2 000 x g and resuspend the pelleted spheroplasts in 180yl EDTA 196 Triton X 100 pH 8 supplemented with 20mg ml lysozyme or 0 2mg ml Lysis Buffer GL and 25yl Prote
5. 0 C through and reuse Collection Tube e Add 600yl Wash Buffer GW2 to the column and centrifuge for 1 min at 11 000 Sample preparation x g Discard flow through and reuse Collection Tube 11 Human or animal tissue 7 Dry silica membrane Cut 25mg of tissue into small pieces Place the sample in a 1 5ml microcentrifuge Centrifuge 1 min at 11 000 x g to remove residual ethanol Place the ISOLATE Il tube proceed to step 2 Genomic DNA Spin Column in a 1 5ml microcentrifuge tube not supplied Note Samples that are difficult to lyse can be ground under liquid nitrogen or may be treated in 8 Elute DNA 1 2 a mechanical homogenizer Add 25mg of tissue to a 1 5ml microcentrifuge tube not supplied add 50 75ul PBS and homogenize Cultured cells Resuspend up to 10 cells in a final volume of 200u Lysis Buffer GL Add 25l Proteinase K solution and 200ul Lysis Buffer G3 Incubate the sample at 70 C for 10 15 min proceed to step 4 Pre lysis Add 180ul Lysis Buffer GL and 25yl Proteinase K solution completely cover sample with solution and vortex Note If processing several samples Proteinase K and Lysis Buffer GL may be premixed directly before use no more than 10 15 min before addition to the sample as Proteinase K will self digest in Lysis Buffer GL without substrate Incubate at 56 C for 1 3 hours until completely lysed shake or vortex occasionally Note Samples can be incubated overnight If RNA free DNA is needed for
6. ISOLATE Il Genomic DNA Kit Product Manual BIOLINE A Meridian Life Science Company Genomic DNA Kit ISOLATE Il Genomic DNA Kit ISOLATE Il Genomic DNA Kit 1 Kit contents 04 2 Description 04 3 Storage 05 4 Safety information 05 5 Product specification 05 6 Equipment and reagents to be supplied by the user 07 7 Important notes 07 tA Buffer preparation and parameters 07 8 Standard protocol 08 8 1 Purifying total DNA from cultured cells and human or 08 animal tissue 9 Alternative protocols 09 9 1 Mouse or rat tails 09 9 2 Bacteria 10 9 3 Yeast 11 9 4 Dried blood spots 11 9 5 Genomic viral DNA from blood 12 9 6 Hair roots 12 9 7 Paraffin embedded tissue 13 9 8 Genomic DNA from fecal material 13 9 9 Viral DNA from fecal material 14 9 10 Bacterial DNA from urine 16 9 11 Viral DNA from urine 16 9 12 Bacterial DNA from cultures clinical specimens or 17 biological fluids 9 13 Detection of Mycobacterium tuberculosis or Legionella 18 pneumophila in respiratory samples 9 14 Detection of Enterohemorrhagic E coli in food 19 9 15 Genomic DNA from dental swabs 19 9 16 Genomic DNA from buccal swabs 20 9 17 Genomic DNA from insects 21 9 18 Genomic DNA from semen 22 10 Troubleshooting guide 24 A Technical support 26 B Ordering information 26 C Associated products 26 D Product warranty and disclaimer 26 2 Product Manual www bioline com isolate 3 1 KIT CONTENTS COMPONENT 10 Pr
7. Proteinase K are prepared see section 7 1 Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C Sample preparation Homogenize a maximum of 50mg insect material under liquid nitrogen Transfer powdered material into a 1 5ml microcentrifuge tube not provided Proceed to step 2 pre lysis of the standard protocol see section 8 1 20 Product Manual www bioline com isolate 9 18 GENOMIC DNA FROM SEMEN To obtain optimal results a differential extraction method is required in order to separate spermatozoa from other cell types such as epithelial cells and or blood Additional reagents needed 8 e Buffer GuEX 200ml o Mix 2ml sterile 5M Guanidine hydrochloride solution should not be autoclaved 2 1ml 1M Tris Cl pH 8 solution 1 05ml 2M NaCl solution 4 2ml 0 5M EDTA solution and 0 2ml 1M NaOH solution o Add water to a volume of 200ml The pH should be between 8 8 5 e sopropanol Before you start e Prepare buffer GuEX e Make sure Lysis Buffer G3 Wash Buffer G2 and Proteinase K are prepared see 2 section 7 1 e Preheat Elution Buffer G to 70 C Sample preparation 10 Transfer sample to a 1 5ml microcentrifuge tube Add 950ul buffer GuEX and 50yl Proteinase K solution Incubate no longer than 15 min at 37 C 11 Separate sample Centrifuge mixture for 4 min at 12 000 x g at room temperature The pellet contains sperm cells sample A pellet Free DNA from epithelial cells and leukocytes is in the
8. contents of Buffer G1 to Buffer G2 Mix well and label Lysis Buffer G3 on the bottle Note The resulting Lysis Buffer G3 is stable for up to one year at room temperature If a white precipitate forms in Lysis Buffer G3 at any time re dissolve by incubating the bottle at 70 C before use Preparing Wash Buffer GW2 Add 96 100 ethanol to Wash Buffer GW2 Concentrate 16ml for the 10 prep kit 28ml for the 50 prep kit and 160ml x 2 for the 250 prep kit Note Mark bottle label to indicate ethanol was added Store Wash Buffer GW2 at room temperature for up to 1 year Preparing Proteinase K Buffer PR Add Proteinase K Buffer PR to the lyophilized Proteinase K 260yl for the 10 prep kit 1 35ml for the 50 prep kit and 3 35ml x 2 for the 250 prep kit Note Proteinase K solution is stable at 20 C for up to 6 months Elution parameters It is possible to modify the elution protocol to improve yield and concentration Use Elution Buffer G preheated to 70 C for one of the following procedures e High yield Two elution steps with 100ul Elution Buffer G to increase yield to 90 100 e High concentration One elution step with 60yl Elution Buffer G to increase concentration by about 13096 Maximal yield 8096 6 Product Manual www bioline com isolate m Genomic DNA Kit e High yield and high concentration Two elution steps Add 50ul Elution Buffer G 3 incubate for 3 min and centrifuge repeat with a second 50ul Elution Buffer G Y
9. downstream applications an RNase digest may be performed RNase not included Add 100ul preheated Elution Buffer G 70 C directly onto the silica membrane Incubate at room temperature for 1 min Centrifuge 1 min at 11 000 x g Note For alternative elution procedures see section 7 1 9 ALTERNATIVE PROTOCOLS 9 1 MOUSE OR RAT TAILS Before you start 1 Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C Sample preparation Cut two 0 6cm pieces of mouse tail and place in a 1 5ml centrifuge tube not supplied Note For rat tails one 0 6cm piece is sufficient 8 Product Manual www bioline com isolate m Genomic DNA Kit 9 3 YEAST Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see 2 Pre lysis Add 180ul Lysis Buffer GL and 25yl Proteinase K solution Completely cover sample with solution and vortex Note If processing several samples Proteinase K and Lysis Buffer GL may be premixed directly section 7 1 before use no more than 10 15 min before addition to the sample as Proteinase K will self digest e Make up sorbitol buffer 1 2M sorbitol 10mM CaCl 0 1M Tris HCl pH 7 5 35mM in Lysis Buffer GL without substrate B mercaptoethanol not supplied Incubate at 56 C overnight or until completely lysed shaking or vortexing i j e Check
10. e Add 500yl Wash Buffer GW2 including ethanol to the spin column and centrifuge 1 min at 6 000 x g RT Discard the flow through Repeat this wash step and discard flow through Dry silica membrane Centrifuge 2 min at 6 000 x g RT to remove Wash Buffer GW2 completely Elute DNA Place the ISOLATE Il Genomic DNA Spin Column in a clean 1 5ml centrifuge tube and elute the DNA with 100 200yl preheated Elution Buffer G 70 C After 2 min incubation centrifuge for 1 min at 6 000 x g RT 22 Product Manual www bioline com isolate 23 10 TROUBLESHOOTING GUIDE LOW DNA YIELD POSSIBLE CAUSE RECOMMENDED SOLUTION Incomplete cell lysis Sample must be vortexed vigorously immediately after addition of Lysis Buffer GL Proteinase K solution Proteinase K digestion not optimal never add Proteinase K directly to Lysis Buffer GL Store dissolved Proteinase K at 20 C for up to 6 months Reagents not applied correctly Prepare buffers and Proteinase K solution according to instructions section 7 1 Reagents not stored optimally store Proteinase K solution at 20 C Store all other components at room temperature Keep bottles tightly closed to prevent evaporation or contamination Suboptimal elution from the column POOR DNA QUALITY POSSIBLE CAUSE Apply preheated 70 C Elution Buffer G directly onto the center of the silica membrane If not using Elution Buffer G make sure elution buffer used is
11. e genomic DNA from any tissue cells bacteria yeast forensic samples serum plasma or other body fluids The hands on time is 20 min for 4 6 preps following the lysis steps The isolated DNA is of high purity A A ratio 1 7 1 9 with yields of 20 35ug see below The ISOLATE Il Genomic DNA Kit is a simple reliable and fast method for isolation of high quality genomic DNA from a variety of sample sources Biological samples are first lysed in chaotropic salt ions in the presence of Proteinase K Ethanol is added to the sample and then processed through a genomic DNA mini spin column containing a silica membrane to which the genomic DNA binds Contaminants and impurities such as salts metabolites and cellular components are effectively removed by simple washing steps with two different buffers High quality purified genomic DNA is then eluted in an elution buffer Please read this manual carefully to familiarize yourself with the ISOLATE Il Genomic DNA protocol before starting also available on www bioline com More experienced users can refer to the bench top protocol for quick referencing during the procedure ISOLATE II GENOMIC DNA COLUMN SPECIFICATIONS Max binding capacity 60ug DNA PN ed born 1 7 1 9 Typical yield 20 35ug Elution volume 60 100yl Sample material Tissue 1 25mg Cells 102 107 4 Product Manual www bioline com isolate Genomic DNA Isolation n Sample preparation Place
12. ect any sample from the lids Open the microcentrifuge tubes Depending on the bacterial strains that are to be detected the incubation at 95 C can be omitted Separate lysis solution from dental swabs Place an ISOLATE II Filter not provided into a Collection Tube Cut off the shaft of the dental swab Transfer the dental swab tip and any associated fluid onto the ISOLATE II Filter Centrifuge for 1 min at 11 000 x g Discard the ISOLATE II Filter and swab Transfer as much as possible of the lysate solution to a 1 5ml microcentrifuge tube not provided Proceed to step 3 of the standard protocol see section 8 1 9 16 GENOMIC DNA FROM BUCCAL SWABS Before you start Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C Sample preparation Collect samples with cotton dacron or CEP swabs ensure that food drink is not consumed for at least 30 min prior to sample collection Scrape firmly against the inside of each cheek several times and allow the swabs to air dry Pre lysis Place dry swab material in 2ml microcentrifuge tubes not provided Add 400 600yl PBS and 25ul Proteinase K solution to each swab The volume of PBS is dependent on the type of swab used Cotton Dacron 400l CEP 600uIl Mix by vortexing for 5s repeat and incubate for 10 min at 56 C 2a m Genomic DNA Kit Separate lysis
13. eps 50 Preps 250 Preps ISOLATE Il Genomic DNA Spin Columns gear 10 50 250 Collection Tubes 2ml 20 100 500 Lysis Buffer GL 5ml 20ml 100ml Buffer G1 6ml 12ml 60ml Buffer G2 1 5ml 3ml 15ml Wash Buffer GW1 6ml 30ml 2 x 75ml Wash Buffer GW2 concentrate 4ml 2x 7ml 2 x 40ml Elution Buffer G 3ml 15ml 75ml Proteinase K lyophilized 6mg 30mg 2x 75mg Proteinase K Buffer PR 0 8ml 1 8ml 8ml Labels for Lysis Buffer G3 1 1 1 User Manual 1 1 1 Bench Protocol Sheet i 1 1 Before use add indicated volume of 96 10096 ethanol and mark wash buffer bottle label 2 DESCRIPTION m wm feme Kit 3 STORAGE Dissolved Proteinase K solution is stable at 20 C for up to 6 months All other kit components should be stored at room temperature 18 25 C and are stable for up to 1 year Storage at lower temperatures may cause precipitation of salts in Buffers GL G1 or G3 Incubate bottle at 50 70 C prior to use to dissolve precipitates 4 SAFETY INFORMATION When working with chemicals always wear a suitable lab coat gloves and safety glasses Buffer G1 and Wash Buffer GW1 contain guanidine hydrochloride This chemical is harmful when in skin contact inhaled or ingested For detailed information please consult the material data safety sheets MSDSs available on our website at www bioline com 5 PRODUCT SPECIFICATIONS The ISOLATE Il Genomic DNA Kit is specially designed for the rapid and efficient isolation of extremely pur
14. equal volume N acetylcysteine NaOH Mix by gently vortexing Incubate mixture for 25 min at room temperature with shaking Adjust volume to 25ml with sterile water Centrifuge for 30 min at 4 000 x g Discard supernatant Resuspend pellet in 0 5 1ml Lysis Buffer GL depending on sample viscosity Transfer 200ul of resuspended sample to a new 1 5ml microcentrifuge tube not provided Proceed to step 2 pre lysis of the standard protocol see section 8 1 9 14 DETECTION OF ENTEROHEMORRHAGIC E COLI IN FOOD This protocol is suited for the selective enrichment and isolation of genomic DNA from enterohemorrhagic E coli EHEC including serotype 0157 H7 in foodstuffs such as fresh cow s milk Before you start Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 Prepare Modified Tryptic Soy Broth mTSB medium 30g Tryptic Soy Broth Gibco 1 5g bile salts No 3 Oxoid 1 5g KH2PO4 Add 900ml water Filter the medium and adjust to pH 7 4 with 2M NaOH Add water to 1L Autoclave at 121 C for 15 min Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C Make a 3 2M solution of sodium acetate Sample preparation Add 225ml prewarmed 37 C mTSB medium supplemented with Novobiocin and 25ml milk in a sterile 1L flask Incubate the mixture in a shaking water bath for 5 6h or overnight at 37 C Centrifuge 100ml culture for 40 min at 6 000 x g Decant the supernatant carefully Resu
15. erase 250 Units BIO 21111 SensiFAST SYBR No ROX Kit 200 Reactions BIO 98002 D PRODUCT WARRANTY AND DISCLAIMER Bioline warrants that its products will conform to the standards stated in its product specification sheets in effect at the time of shipment Bioline will replace free of charge any product that does not conform to the specifications This warranty limits Bioline s liability only to the replacement of the product BIOLINE A Meridian Life Science Company www bioline com United Kingdom Bioline Reagents Ltd 16 The Edge Business Centre Humber Road London NW2 6EW Tel 44 0 20 8830 5300 Fax 44 0 20 8452 2822 email info uk bioline com Bioline GmbH Im Biotechnologiepark TGZ 2 D 14943 Luckenwalde Tel 49 0 3371 68 12 29 Fax 49 0 3371 68 12 44 email info de bioline com Bioline Aust Pty Ltd PO Box 122 Alexandria NSW 1435 Tel 61 0 2 9209 4180 Fax 61 0 2 9209 4763 email info aust bioline com Bioline USA Inc 305 Constitution Drive Taunton MA 02780 Tel 1 508 880 8990 Fax 1 508 880 8993 Order Toll Free 1 888 257 5155 email info us bioline com 26 Product Manual www bioline com isolate PM1013V1 1WEB
16. ield 85 100 at a high concentration Lyse sample Vortex sample briefly and add 200ul Lysis Buffer G3 Vortex vigorously and incubate at 70 C for 10 min Note If insoluble particles are visible centrifuge for 5 min at high speed and transfer the supernatant to a new microcentrifuge tube Adjust DNA binding conditions Vortex briefly and add 210ul ethanol 96 100 to the sample Vortex vigorously Note After addition of ethanol a stringy precipitate may become visible This will not affect the DNA isolation 5 Bind DNA For each sample place an ISOLATE II Genomic DNA Spin Column into a Collection Tube Add all of the sample to the column and centrifuge for 1 min at 11 000 x g Discard the flow through and reuse Collection Tube Repeat at a higher g force if samples are not completely filtered through matrix DNA Storage Store isolated DNA at 20 C Several freeze thaw cycles will not interfere with most 4 downstream applications however for long range PCR or high sensitivity especially in real time PCR store in aliquots to avoid multiple freeze thawing 8 STANDARD PROTOCOL 8 1 PURIFYING DNA FROM CULTURED CELLS AND HUMAN OR ANIMAL TISSUE Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 6 Wash silica membrane e Set an incubator or water bath to 56 C e Add 500yl Wash Buffer GW1 Centrifuge for 1 min at 11 000 x g Discard flow e Preheat Elution Buffer G to 7
17. inase K solution and vortex vigorously Incubate at lysostaphin not supplied 56 C for 1 3 hours until completely lysed shaking or vortexing occasionally Note Samples can be incubated overnight If RNA free DNA is needed for downstream b Set an incubator or water bath to 56 C applications an RNase digest may be performed RNase not included e Preheat Elution Buffer G to 70 C Proceed with step 3 of the standard protocol see section 8 1 1 Sample preparation 9 4 DRIED BLOOD SPOTS Up to 1ml of bacterial culture can be used for the preparation depending on density Before you start culture medium bacterial strain etc e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see Centrifuge up to 1ml culture for 5 min at 8 000 x g Remove supernatant section 7 1 2 Pre lysis e Set an incubator or water bath to 56 C and 94 C Resuspend pellet in 180ul Lysis Buffer GL and 25yl Proteinase K solution and vortex e Preheat Elution Buffer G to 70 C vigorously Incubate at 56 C for 1 3 hours until completely lysed shake or vortex occasionally 1 Sample preparation ieee ca s dou Doe d BA a Ae ae dec for downstream Cut out one or two dried blood spots 15 and 30mm in area as accurately as possible Note For hard to lyse bacteria such as Gram positive bacteria a preincubation is necessary Cut spots into small pieces and place into a 1 5ml microcentrifuge tube not supplied Resuspend the pelleted cells in a
18. lied Elute DNA Add 100ul pre warmed Elution Buffer G 70 C to the column Incubate for 3 5 min at 70 C with the lid closed Centrifuge for 1 min at 4 500 x g For alternative elution procedures see section 7 1 PCR Use 10ul purified DNA as template in a 20ul PCR reaction Add an inhibition control mix 10jl purified DNA template with human DNA Amplify with primers specific for a human DNA sequence such as B actin B globin or another reference gene of choice 9 10 BACTERIAL DNA FROM URINE This protocol is designed for purification of bacterial genomic DNA from urine samples Before you start Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C 14 Product Manual www bioline com isolate Sample preparation Centrifuge a 1ml urine sample at 13 000 x g for 30 min Discard the supernatant Add another 1ml of the urine sample to the pelleted material Centrifuge at 13 000 x g for 30 min and discard supernatant Repeat the centrifugation with a third 1ml sample of urine and discard the supernatant Note Fresh urine samples should be used Storage at 20 C to 80 C is only recommended for 1 2 days After thawing incubate the sample at 40 C until all precipitates are dissolved when stored at low temperatures urine tends to form precipitates Proceed to step 2 pre lysis of the standard protocol see sectio
19. lysis buffer instead of Lysis Buffer GL see above supplemented with lysozyme or lysostaphin and incubate for 30 60 min at 37 C Add 25ul Proteinase K incubate at 56 C until complete lysis is obtained Proceed with step 3 of the standard protocol see section 8 1 10 Product Manual www bioline com isolate 11 2 Pre lysis Add 180ul Lysis Buffer GL and incubate at 94 C for 10 min Cool and add 25yl Proteinase K solution Completely cover sample and incubate at 56 C for 60 min shaking or vortexing occasionally 3 Lyse sample Vortex sample briefly and add 200ul Lysis Buffer G3 Vortex vigorously Proceed with step 4 of the standard protocol see section 8 1 9 5 GENOMIC VIRAL DNA FROM BLOOD Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 e Preheat Elution Buffer G to 70 C 1 Lyse blood Add 25yl Proteinase K Buffer PR and 200yl sample into a 1 5m microcentrifuge tube not supplied Note Make up sample to 200 with PBS if using less volume For cultured cells resuspend up to 5 x 108 cells in 200ul PBS Add 200ul Lysis Buffer G3 and vortex vigorously for 10 20s Incubate samples at 70 C for 10 15 min Note The lysate should turn brownish during incubation with Lysis Buffer G3 If processing older or clotted blood increase Proteinase K incubation time up to 30 min and vortex vigorously several times during incubation 2 Adjust DNA binding condition
20. n 8 1 9 11 VIRAL DNA FROM URINE This protocol is designed for purification of viral genomic DNA from urine samples Before you start Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C Sample preparation Note If frozen urine samples are used precipitates may appear after thawing and must be dissolved before centrifugation Incubate sample at 37 40 C for 30 min If precipitates do not dissolve allow precipitate to sediment and perform this step with only the supernatant Centrifuge aliquots of the urine sample for 10 min at full speed e g 4ml 4 x 1ml ina 1 5ml microcentrifuge tube Carefully decant the supernatant Pre lysis Resuspend the pellet in Lysis Buffer GL and Proteinase K Follow either procedure A or B A Resuspend first pellet in 180ul Lysis Buffer GL and 25yl Proteinase K Transfer the resuspended solution from tube 1 to the tube containing the pellet from the second 1ml aliquot Resuspend this pellet Repeat for the pellets from aliquots 3 and 4 The tube should now contain a resuspensed solution of all four pellets Proceed with Step 3 Or B Resuspend each pellet individually with 180pl Lysis Buffer GL and 25ul Proteinase K Pool all four resuspended pellets together into one tube Proceed to step 3 Note With this procedure at step 5 the ISOLATE Il Genomic DNA Spin Column will need
21. on Trim excess paraffin off the block and cut small sections up to 25mg With tweezers or toothpicks place the sections into microcentrifuge tubes not supplied Add 1ml n octane or xylene to each tube and vortex vigorously Incubate at room temperature for 30 min vortexing occasionally Centrifuge at 11 000 x g for 3 min Discard supernatant Add 1ml ethanol 96 100 and mix by inverting several times Centrifuge at 11 000 x g for 3 min Discard supernatant Repeat the ethanol washing step Remove as much of the ethanol as possible Incubate the open tube at 37 C until the ethanol has evaporated 15 min Proceed with step 2 of the standard protocol see section 8 1 9 8 GENOMIC DNA FROM FECAL MATERIAL This protocol is suited for the isolation of genomic DNA from fecal material Whilst this protocol is optimized for human cells and microorganisms a supplementary protocol for viral DNA is also provided see section 9 9 Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 12 Product Manual www bioline com isolate m Genomic DNA Kit e Set an incubator or water bath to 37 C and 56 C 4 Adjust DNA binding conditions e Preheat Elution Buffer G to 70 C Sample preparation Add 250mg fecal material to 1ml TE buffer Vortex vigorously 30s to resuspend the sample Centrifuge for 15 min at 4 000 x g Remove the supernatant Resuspend the pellet in 0 2 1ml L
22. on according to instructions section 7 1 Ensure ethanol is added to lysates before loading on columns SUBOPTIMAL PERFORMANCE OF EXTRACTED GENOMIC DNA IN ENZYMATIC REACTIONS RECOMMENDED SOLUTION Ethanol carry over Be sure to remove all traces of Wash Buffer GW2 before eluting the DNA If necessary repeat silica membrane drying step a second time Do not chill Wash Buffer GW2 before use Cold buffer will not remove salt effectively Equilibrate Wash Buffer GW2 to room temperature before use Contamination of DNA with inhibitory substances We recommend elution with Elution Buffer G as chemicals such as EDTA that are found in other buffers can interfere with downstream applications If the AX A ratio of the eluate is below 1 6 repeat the purification procedure Add equal volumes of Lysis Buffer G3 and ethanol to the eluate load column and proceed with step 3 of the protocol 24 Product Manual www bioline com isolate 25 A TECHNICAL SUPPORT For technical assistance or more information on these products please email us at tech bioline com B ORDERING INFORMATION PRODUCT PACK SIZE CAT NO ISOLATE Il Genomic DNA Kit 10 Preps BIO 52065 ISOLATE Il Genomic DNA Kit 50 Preps BIO 52066 ISOLATE Il Genomic DNA Kit 250 Preps BIO 52067 C ASSOCIATED PRODUCTS PRODUCT PACK SIZE CAT NO ISOLATE II Blood DNA Kit 50 Preps BIO 52063 ISOLATE II Plant DNA Kit 50 Preps BIO 52069 MyTaq HS DNA Polym
23. s Add 210ul ethanol 96 10096 and vortex 3 Bind DNA For each preparation place one ISOLATE Il Genomic DNA Spin Column in a Collection Tube and load the sample onto the column Ensure all lysate is loaded Centrifuge for 1 min at 11 000 x g Repeat at a higher g force if samples are not completely filtered through matrix Place column in a new Collection Tube 2ml Proceed with step 6 of the standard protocol see section 8 1 9 6 HAIR ROOTS Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 e Set an incubator or water bath to 56 C e Preheat Elution Buffer G to 70 C m Genomic DNA Kit 1 Sample preparation Cut up to 100 hair roots from the hair sample and place in a 1 5ml centrifuge tube not supplied 2 Pre lysis Add 180ul Lysis Buffer GL and freeze the samples in liquid nitrogen Thaw samples in a 56 C water bath Repeat this freeze thawing procedure 4 times Add 25ul Proteinase K solution and incubate at 56 C overnight or until completely lysed shake or vortex occasionally Proceed with step 3 of the standard protocol see section 8 1 9 7 PARAFFIN EMBEDDED TISSUE Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 e Check that n octane or xylene not supplied is available e Set an incubator or water bath to 37 C and 56 C e Preheat Elution Buffer G to 70 C 1 Sample preparati
24. spend the pellet in 2ml sterile water Centrifuge for 10 min at 10 000 x g Pre lysis Resuspend the pellet in 180ul Lysis Buffer GL and add 25yl Proteinase K solution Perform the standard protocol from step 3 lyse sample see section 8 1 Post elution After elution perform an ethanol precipitation of the DNA as follows Add 20yl 3 2M sodium acetate and 400yl 96 100 ethanol to 200l eluate Centrifuge for 30 min at 11 000 x g Discard the supernatant and wash the pellet with 1ml 70 ethanol Resuspend the pellet in 10pl sterile water 9 15 GENOMIC DNA FROM DENTAL SWABS Before you start Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C Sample preparation Place swab material e g foam cotton paper brushes plastic in a 1 5ml microcentrifuge tube not provided 18 Product Manual www bioline com isolate 2a Pre lysis Add 180ul Lysis Buffer GL and 25yl Proteinase K to each sample Close the microcentrifuge tube lid and spin briefly for 15s at 1 500 x g in order to submerge swab material Incubate at room temperature for 5 min Vortex the tube vigorously for 15 s and spin briefly for 15s at 1 500 x g Incubate the tubes at 70 C in an incubator for 10 min Place a weight on top of the tubes to stop the caps from opening Increase the temperature to 95 C for 5 min Spin briefly for 15s at 1 500 x g to coll
25. supernatant sample B supernatant Remove supernatant Carefully remove the supernatant sample B supernatant Transfer to a fresh tube and process separately see step 6 Add buffer to pellet Add 700ul buffer GuEX to the pellet sample A pellet centrifuge for 4 min at 12 000 x g and discard the supernatant Repeat this wash step 2 3 times Resuspend pellet Resuspend sample A pellet in a minimum of 300l Buffer GL Lyse sample o Sample A pellet Add 25yl Proteinase K stock solution mix by vortexing and incubate overnight at 60 65 C o Sample B supernatant Add 10yl Proteinase K stock solution mix by vortexing and incubate overnight at 60 65 C m Genomic DNA Kit Clarify sample Centrifuge samples for 5 min at 12 000 x g at room temperature RT in order to remove any unsoluble cell material Proceed with the clear supernatant Bind DNA o Sample A pellet Add 300yl Lysis Buffer G3 and 300yl isopropanol to the clear supernatant and apply the sample successively to the ISOLATE II Genomic DNA Spin Column Centrifuge 1 min at 6 000 x g RT If the sample is not passing through the membrane completely repeat centrifugation step o Sample B supernatant Add 400ul of isopropanol to the clear supernatant and apply the sample successively to the ISOLATE Il Genomic DNA Spin Column Centrifuge 1 min at 6 000 x g RT If the sample is not passing through the membrane completely repeat centrifugation step Wash silica membran
26. to be loaded centrifuged in successive steps due to the increased lysate volume m Genomic DNA Kit 3 Lyse sample Add 2003 Lysis Buffer G3 vortex and incubate for a minimum of 20 min at 70 C 4 Adjust DNA binding conditions Add 210ul ethanol 96 100 to sample and vortex vigorously 5 Bind DNA Apply sample to an ISOLATE Il Genomic DNA Spin Column placed in a Collection Tube Centrifuge 1 min at 4 500 x g Discard flow through and return the column to Collection Tube 6 Washsilica membrane Add 500yl Wash Buffer GW1 to the column Centrifuge for 1 min at 4 500 x g Discard flow through and return the column to Collection Tube Add 600yl Wash Buffer GW2 to the column Centrifuge for 2 min at 11 000 x g Discard the flow through and return the column to the Collection Tube 7 Dry silica membrane Open the lid of the ISOLATE Il Genomic DNA Spin Column and incubate for 1 2 min at 70 C Residual ethanol is removed during this step 8 Elute DNA Add 70ul pre warmed 70 C Elution Buffer G to the column Close the lid and incubate for an additional 3 5 min at 70 C Centrifuge for 1 min at 4 500 x g 9 12 BACTERIAL DNA FROM CULTURES CLINICAL SPECIMENS OR BIOLOGICAL FLUIDS This protocol is suited for the detection of bacterial genomic DNA from a range of starting materials Before you start e Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 e Set an incubator or water bath to 56 C
27. up to 25mg tissue into 1 5ml tube 3 Sample pre lysis j Add 180yl Lysis Buffer GL 25yl Proteinase K solution J l Vortex Incubate 56 C 1 3 hrs or overnight Vortex Sample lysis Add 200ul Lysis Buffer G3 and vortex Incubate 70 C 10 min Adjust DNA binding conditions Add 210ul ethanol 11 000 x g 1 min 1st and 2 4 11 000 x g 1 min Vortex Bind DNA Load lysate Wash silica membrane 1 wash 500ul Wash Buffer GW1 2nd wash 600ul Wash Buffer GW2 11 000 x g 1 min Dry silica membrane _ 11 000 x g 1 min Elute DNA Add 100yl Elution Buffer G 70 C Incubate RT 1 min Isolated DNA am Genomic DNA Kit 6 EQUIPMENT AND REAGENTS TO BE SUPPLIED BY USER When working with chemicals always wear a suitable lab coat protective goggles and disposable gloves e 96 100 ethanol for Wash Buffer GW2 e Microcentrifuge tubes 1 5ml e Sterile DNase free tips e Pipettes e Microcentrifuge capable of 11 000 x g e Vortex mixer e Thermal heating block e Equipment for sample disruption and homogenization e Personal protection equipment lab coat gloves goggles Molecular biology grade ethanol is recommended Do not use denatured alcohol which contains unwanted additives such as methanol and acetone 7 I IMPORTANT NOTES 7 1 BUFFER PREPARATION AND PARAMETERS Preparing Lysis Buffer G3 Transfer the
28. upernatant Add 400yl Lysis Buffer GL then 35yl Proteinase K and mix by vortexing Transfer the supernatant into a sterile 1 5ml microcentrifuge tube not supplied Lyse sample Add 400ul Lysis Buffer G3 to the supernatant from step 2 and mix by vortexing Incubate for a minimum of 30 min at 70 C Add 420ul ethanol 96 100 to the lysed sample from step 3 and mix by vortexing Bind DNA For each sample place one ISOLATE Il Genomic DNA Spin Column into a Collection Tube and load the lysate Centrifuge for 1 min at 4 500 x g Discard the flow through and return column into Collection Tube Repeat lysate loading to the column and centrifugation steps if necessary If sample does not completely transfer through the silica membrane repeat centrifugation step at 11 000 x g Discard flow through Wash silica membrane e Add 600yl Wash Buffer GW1 to the column Centrifuge for 1 min at 4 500 x g Discard the flow through and reuse Collection Tube e Add 600yl Wash Buffer GW2 to the column Centrifuge for 1 min at 4 500 x g Discard the flow through and reuse Collection Tube e Add 600u Wash Buffer GW2 to the column Centrifuge for 2 min at 11 000 x g Discard the flow through Place ISOLATE Il Genomic DNA Spin Column into a new Collection Tube Dry silica membrane Incubate ISOLATE Il Genomic DNA Spin Column with the lid opened for 1 2 min at 70 C to remove residual ethanol Place the column in a new 1 5ml microcentrifuge tube not supp
29. ysis Buffer GL Add sufficient buffer to thoroughly resuspend the sample Transfer 200ul of the resuspended sample to a new microcentrifuge tube and add 25ul of Proteinase K Incubate for 1 3 hours at 56 C Note Cells from human bacterial and pathogenic origin are found in fecal material and will lyse during the Proteinase K Lysis Buffer GL incubation at 56 C with different efficiencies To detect cells that are difficult to lyse e g some bacteria and parasites performing an additional incubation at increased incubation temperature up to 95 C 5 10 min may help to increase DNA yield Proceed to step 3 lyse sample of the standard protocol see section 8 1 Release of bacterial pathogen DNA can be monitored by using qPCR or a similar technique to examine the human non human ratio 9 9 VIRAL DNA FROM FECAL MATERIAL This protocol is suited for the isolation of viral genomic DNA from fecal material Before you start Make sure Lysis Buffer G3 Wash Buffer GW2 and Proteinase K are prepared see section 7 1 Set an incubator or water bath to 56 C Preheat Elution Buffer G to 70 C Prepare 0 9 w v NaCl in molecular biology grade water Sample preparation Suspend the fecal sample approx 0 5g in 0 9 NaCl solution max 4ml Centrifuge the fecal sample 5 min at 800 x g at room temperature Filter the supernatant using a 0 22 0 45um sterile filter Centrifuge for 1 min at 11 000 x g Pre lysis Carefully decant the s
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