Invisorb Universal Bacteria HTS 96 Kit/ C User manual
Contents
1. Add 90 ml 99 7 Isopropanol to the Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 80 ml 96 100 ethanol to the bottle Wash Buffer I Add 140 ml 96 100 ethanol to the bottle Wash Buffer II Add 8 5 ml ddH20O to the Proteinase K Mix thoroughly and store at 20 C Add provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly Aliquot and store at 20 C 100 ul Lysozyme mixture sample 24x 96 DNA extractions Add 390 ml 99 7 Isopropanol to the Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 450 ml 96 100 ethanol to the bottle Wash Buffer Add 630 ml 96 100 ethanol to the bottle Wash Buffer II Add 50 ml ddH20 to the Proteinase K Mix thoroughly and store at 20 C Add provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly Aliquot and store at 20 C 100 pl Lysozyme mixture sample 1 Adjusting the thermomixer water bath incubation oven or others to 65 C 2 To increase the yield warming up the needed amount of Elution Buffer to 65 C per sample 1x 100 ul Elution Buffer are needed 16 Invisorb Universal Bacterial HTS 96 Kit C 0413 Equipment and reagents to be supplied by user When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Fo2. 96 well format using acentrifuge CE stratecee molecular User manual Invisorb Universal Bacteria HTS 96 Kit C for purification of bacterial or parasite DNA from different kinds of starting materials ina 7033300x00 gaali STRATEC Molecular GmbH D 13125 Berlin Fr MUTUATOATOTAATOAVOOVOOTOATOVOQTOOVOOTOVIOVOOVNOVOOTOVNOTONITOOVTOATOVNOTNOVONTD Invisorb Universal Bacteria HTS 96 Kit The Invisorb Universal Bacteria HTS 96 Kit is the ideal tool in convenient 96 well format for isolation and purification of bacterial DNA from different clinical specimen e g whole blood swabs biopsy material and from cell free body fluids e g urine serum plasma and synovial fluids Invisorb Universal Bacteria HTS 96 Kit suits to manual and automated use Ultra pure genomic DNA gets purified for enhanced performance in sensitive applications Additionally yields of the ready to use DNA are reproducible Large sample numbers in this 96 well format are handled time saving The user can be flexible in his choices in the range of starting material No cross contamination will occur The kit is neither validated for the isolation of human or bacterial genomic DNA from faecal samples nor for viral nucleic acids or RNA isolation Trademarks Invisorb VACUUBRAND are registered marks trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law The Invis
3. cool down and place plate in same orientation back continuously shaking of the lysates during lysis will improve lysis efficiency homogenizing or sample crushing will improve lysis efficiency use proper volume of Lysis Buffer HL and Binding Buffer HL mix sample thoroughly with Binding Buffer HL by pipetting up and down prior to transfer the sample onto the DNA Binding Plate B make sure that the correct amount of ethanol is added to the Wash Buffers and stored correctly increase centrifugation time in step 15 increase incubation time with pre warmed Elution Buffer to 10 min pre warm Elution Buffer up to 80 C use higher volume of Elution Buffer eluting DNA with lower amount of Elution Buffer eluted DNA is colored insufficient washing wash again with Wash Buffer II problems with eluat contains verify centrifugation time and speed step 15 If subsequent ethanol necessary increase centrifugation time to remove applications e g ethanol with PCR eluat contains salt Wash Buffer should have room temperature Wash Buffer shouldn t contain any precipitates if there are any precipitates solve them by careful too much too low warming up to room temperature t late DNA esa os SER optimized needed amount of template DNA DNA is contaminated perform RNAse digestion with RNA Reference Sambrock J Fritsch E F and Maniatis T 1989 Molecular cloning A laboratory manual Cold Spring Harbor Labora
4. E mail info berlin stratec com www stratec com 1C31C 04 2013
5. mean value and the standard deviation for the samples in Fig 2 the results are listed below Colour Name Rep Ct Rep Ct Std Dev HH pos 26 46 0 95 Hl neg 33 95 1 75 HH etc 16 4 0 03 Monte 36 11 0 39 8 Invisorb Universal Bacterial HTS 96 Kit C 0413 Reproducibility To show the reproducibility of the Invisorb Universal Bacteria HTS 96 Kit different bacterial DNA extractions were done with dilution series Typical results are shown below a x 10 dD Ti Tr ann z ur t Fx 6o Fig 3 On the left side real time PCR result of an extracted DNA from a dilution series of B subtilis cells in TE buffer On the right side the melting curve for the resulting PCR products Tab 2 Ct values and standard deviations for the real time PCR shown above in figure 4 Colors belongs to the curves in figure 3 Colour Name mean Ct mean Ct Std Dev HH os 17 63 0 09 DY 100 4 20 92 I 10e 5 23 63 0 36 L 10e 6 27 03 1 08 t0e 7 35 58 4 57 HH Pre 14 39 0 14 Monte 3530 9 Invisorb Universal Bacterial HTS 96 Kit C 0413 Influence of different matrices To shown the usability of different clinical relevant matrices the same amount of B subtilis cell were spiked to 100uL of whole blood plasma urine and TE buffer and the DNA extracted following the kit Resulting DNA were analyzed
6. speed for 10 min to dry the membrane discard 2 ml Collection Plate place the DNA Binding Plate B on the top of a Elution Plate L add prewarmed 100 ul Elution Buffer per cavity incubate 2 min at RT centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 3 min 18 Invisorb Universal Bacterial HTS 96 Kit C 0413 Protocol 1 Purification of bacterial DNA from clinical samples serum plasma blood and cell free body fluid sample using a centrifuge Please read the protocols prior to the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer HL see page 16 Sample Preparation Use up to 100 ul whole blood plasma or other cell free body fluid It is recommended for smaller sample volumes than 100 ul to adjust it to 100 ul with 1x PBS or water before starting the purification and isolation procedure If sample are very viscous take care not to clog the membrane with this samples In cases of very viscous samples use smaller sample volumes and adjust to 100 ul with water or 1x PBS 1 Transfer the adjusted sample to 100 ul into one well of the 2 0 ml Collection Plate and proceed the purification on the Centrifuge 2 Add 100 ul diluted Lysozyme to each well of the 2 ml Collection Plate and incubate for 10 min at RT 3 Add 20 ul of prior diluted Proteinase K to each well of the 2 ml Collection Plate vortex shortly for stirring therefore cover
7. the 2 ml Collection Plate Place the DNA Binding Plate B on top of a Elution Plate L Add 100 ul Elution Buffer prewarmed to 65 C directly onto the membrane in each well and incubate for 5 min at ambient temperature Cover the DNA Binding Plate B with the Plate Lid and place the whole block DNA Binding Plate B Elution Plate L in the rotor bucket of the centrifuge Centrifuge for 3 min at 1 400 1 700 x g 3 700 4 000 rpm Take the DNA Binding Plate B and the Elution Plate L out of the centrifuge very carefully in order to avoid cross contaminations with adherent fluid Discard the DNA Binding Plate B Seal the Elution Plate L with a Sealing Foil for a sure storage of the DNA samples 20 Invisorb Universal Bacterial HTS 96 Kit C 0413 Protocol 2 Purification of bacterial DNA from bacterial cultures using a centrifuge Please read the protocols prior to the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer HL see page 16 Sample Preparation For isolation of DNA from bacteria pellets max 1 x 10 bacteria cells take an aliquot of the bacteria culture and spin down at 9 300 x g 10 000 rpm for 3 min Remove carefully the complete supernatant Normalization Add 100 ul water or 1x PBS to each pellet and resuspend the pellet by pipetting up and down Transfer the resuspended sample into one well of the 2 ml Collection Plate and proceed the purifi
8. the membrane and elimination of ethanol elution of total DNA genomic bacterial mitochondrial ALON gt The samples will be lysed in an optimized buffer and enzyme mixture The lysates are transferred to the subsequent purification procedure based on silica membranes The DNA binds to membrane followed by washing steps and the final elution The purified high quality DNA is ready to use for subsequent downstream applications e g for PCR amplification quantitative PCR real time PCR or other routine diagnostic methods This instruction contains 6 protocols Sampling and storage of starting material Blood buffy coat Blood samples and buffy coat can be stored at room temperature 18 25 C up to 6 hours or at 2 8 C up to 24 hours For long term storage we recommend freezing samples at 20 C or 80 C Multiple thawing and freezing before isolating the DNA should be avoided If cryoprecipitates formed during thawing of frozen samples are visible avoid aspirating them during aspiration of the sample they could clog the Filterplate The amount of purified DNA in the Invisorb Universal Bacteria HTS 96 Kit C procedure from max 100 ul whole blood or 25 ul buffy coat depends on the white blood cell content of each blood sample Various different primary tubes and anticoagulants except heparin can be used to collect blood samples for the Invisorb procedure Biopsy material Best results are obtained with fresh material or mate
9. wells with a sealing foil or mix completely by pipetting up and down Incubate the covered plate at 65 C for about 10 min under continuous shaking 800 rpm 4 Remove the cover and add 200 ul of Lysis Buffer HL to each well vortex shortly for stirring therefore cover wells with a sealing foil or mix completely by pipetting up and down Incubate the covered plate at 65 C for about 3 min under continuous shaking 800 rpm Note Incubation at RT instead of at 65 C during step 3 and 4 may reduce the lysis efficiency for some bacteria 5 Add 200 ul Binding Buffer HL to each well of the 2 ml Collection Plate and mix thoroughly by pipetting up and down Cover the Collection Plate with the Plate Lid Place the 2 ml Collection Plate into a centrifuge and spin shortly centrifuge up to 200 x g 1 000 rpm and stop the centrifugation Remove the cover and place the DNA Binding Plate B on the top of another 2 ml Collection Plate Transfer the suspension completely into each well of the DNA Binding Plate B Cover the DNA Binding Plate B with the Plate Lid Load the whole block DNA Binding Plate B 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 6 min at RT Take the DNA Binding Plate B 2 ml Collection Plate out of the centrifuge Remove the Plate Lid and discard the filtrate Place the DNA Binding Plate B back to the top of the 2 ml Collection P
10. Protocol 6 Purification of bacterial DNA from small biopsy samples 22 Troubleshooting 23 Appendix General notes on handling DNA 24 Ordering information 25 Invisorb Universal Bacterial HTS 96 Kit C 0413 Kit contents of the Invisorb Universal Bacteria HTS 96 Kit C Store all kit components at room temperature Diluted Proteinase K at 20 C Lyophilized Lysozyme should be stored at 20 C Dissolved Lysozyme must be stored in aliquots at 20 C 2x 96 DNA extractions 4x 96 DNA extractions 24x 96 DNA extractions Binding Buffer HL final volume 2 x 32 ml final volume 120 ml Catalogue No 7033300200 7033300300 7033300400 Lysozyme Buffer 25 ml 45 ml 250 ml Lysozyme 250 mg 450 mg 2 59 Proteinase K 3x 1 5 ml 8 5 ml 50 ml Lysis Buffer HL 50 ml 90 ml 500 ml 2x8 ml 30 ml 130 ml final volume 520 ml Wash Buffer 80 ml final volume 160 ml 2 x 80 ml final volume 2 x 160 ml 2 x 450 ml final volume 2 x 900 ml Wash Buffer Il 2x45 ml final volume 2 x 150 ml 3 x60 ml final volume 3 x 200 ml 4x 270 ml final volume 4 x 900 ml Elution Buffer 30 ml 60 ml 250 ml DNA Binding Plate B 2 4 6x4 2 0 ml Collection Plate 4 2x4 12x4 Elution Plate L 2 4 24 Plate Lid 2 4 24 Sealing Foils 4 8 48 Manual 1 1 1 add 390 ml 99 7 lsopropanol to the Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several tim
11. by real time PCR Results are shown below None Fionn AM y D ug one iz ke 40 TE w EE To Fig 4 Real time PCR results from DNA extracted from the same amount of B subtilis cell from different matrices On the left amplification data on the right melting curves for the resulting PCR products Tab 3 Ct values for the extraction of the same amount of B subtlis from different matrices Colour Name Rep Ct Rep Ct Std Dev M TE buffer 10e 5 25 46 1 57 HH biood 100 ul 10e 5 25 30 1 19 Hl blood 50 ut 10e 5 25 60 0 44 Ej plasma 100 uL 10e 5 25 48 0 61 B urine 100 ut 106 5 25 98 0 28 E PTC 16 21 0 07 H NTC 30 50 Tab 4 Ct value and standard deviation over all sample shown in table 3 Rep Ct Rep Ct Std Dev 25 56 0 75 10 Invisorb Universal Bacterial HTS 96 Kit C 0413 Investigation of the recovery of DNA from Staphylococcus aureus To test the kit with other bacteria than B subtilis a test with two different inactivated Staphylococcus aureus were done The Staphylococcus aureus were from a bouillon inactivated with 1 volume of 96 ethanol The concentrations of cells were unknown For the experiment 50 ul and 5 ul were used and filled up to 100 ul with TE buffer The extraction was done following the Manual The detection was done with the SureClin MRSA PLUS V Kit from Congen Biotechnologie GmbH Results are sh
12. cation process Follow the Protocol 1 from step 2 Protocol 3 Purification of bacterial DNA from food samples using a centrifuge Please read the protocols prior to the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer HL see page 16 Sample Preparation 35 LMBG Lebensmittel und Bedarfsgegenstandegesetzes Take 25 g of the food material and homogenize the material Add to the homogenized material 225 ml of the recommended culture media e g Fraser media and cultivate the recommended time 24h Take a 1 ml aliquot of this culture and spin down at 9 300 x g 10 000 rpm for 3 min Remove careful completely the supernatant Normalization Add 100 ul water or 1x PBS to each pellet and resuspend the pellet by pipetting up and down Transfer the resuspended sample into one well of the 2 ml Collection Plate and proceed the purification process Follow the Protocol 1 from step 2 Protocol 4 Purification of bacterial DNA from swab samples using a centrifuge Please read the protocols prior to the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer HL see page 16 Sample Preparation It is recommended to rinse each swab with 500 ul 1x PBS or water and use an 100 ul aliquot of the rinsed water for the extraction of the bacterial DNA If the swab will be delivered in a stabilizati
13. ents of the Invisorb Universal Bacteria HTS 96 Kit except MAP Solution A and Lysozyme should be stored at room temperature and are stable for at least 12 months under these conditions Lysozyme Lyophilized Lysozyme should be stored at 20 C Dissolved Lysozyme must be stored in aliquots at 20 C Avoid repeated freezing and thawing Proteinase K Dissolved Proteinase K must be stored at 20 C Wash Buffer I and II Wash Buffer charged with ethanol should be stored at room temperature and should be appropriate sealed If there are any precipitates within the provided solutions solve these precipitates by careful warming up to room temperature up to 30 C Room temperature is defined as range from 15 30 C Quality control and product warranty STRATEC Molecular warrants the correct function of the Invisorb Universal Bacteria HTS 96 Kit for applications as described in this manual Purchaser must determine the suitability of the Product for its particular use Should any Product fail to perform the applications as described in the manual STRATEC Molecular will check the lot and if STRATEC Molecular investigates a problem in the lot STRATEC Molecular will replace the Product free of charge STRATEC Molecular reserves the right to change alter or modify any Product to enhance its performance and design at any time In accordance with STRATEC Molecular s ISO 9001 2000 and ISO EN 13485 certified Quality Management System the
14. es add 450 ml EtOH to each bottle Wash Buffer add 630 ml EtOH to each bottle Wash Buffer II add 50 ml ddH2O to the tube Proteinase K and store at 20 C add provided amount of Lysozyme to the bottle Lysozyme Buffer and mix it thoroughly Aliquot and store at 20 C add 24 ml 99 7 Isopropanol to each Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 80 ml EtOH to the bottle Wash Buffer add 105 ml EtOH to each bottle Wash Buffer II add 1 5 ml ddH20 to each tube Proteinase K and store at 20 C add provided amount of Lysozyme to the bottle Lysozyme Buffer and mix it thoroughly Aliquot and store at 20 C add 90 ml 99 7 lsopropanol to the Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times add 80 ml EtOH to each bottle Wash Buffer add 140 ml EtOH to each bottle Wash Buffer II add 8 5 ml ddH20 to the tube Proteinase K and store at 20 C add provided amount of Lysozyme to the bottle Lysozyme Buffer and mix it thoroughly Aliquot and store at 20 C Initial Steps Invisorb Universal Bacterial HTS 96 Kit C 0413 Symbols Manufacturer Lot number Catalogue number Date of manufacture Expiry date Consult operating instructions Temperature limitation Do not reuse ox BKE AEE Storage All buffers and kit cont
15. format at www stratec com for each STRATEC Molecular Product and its components If buffer bottles are damaged or leaking WEAR GLOVES AND PROTECTIVE GOGGLES when discarding the bottles in order to avoid any injuries STRATEC Molecular has not tested the liquid waste generated by the Invisorb Universal Bacteria HTS 96 Kit procedure for residual infectious materials Contamination of the liquid waste with residual infectious materials is highly unlikely but cannot be excluded completely Therefore liquid waste must be considered infectious and be handled and discarded according to local safety regulations European Community risk and safety phrases for the components of the Invisorb Universal Bacteria HTS 96 Kit procedure to which they apply are listed below as follows Lysis Buffer HL danger H302 315 319 P280 305 351 338 Proteinase K Wash Buffer I danger waming H315 319 334 335 P280 305 351 338 310 405 H302 312 332 412 EUH032 P273 H302 Harmful if swallowed H315 Causes skin irritation H319 Causes serious eye irritation H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled H335 May cause respiratory irritation H312 Harmful in contact with skin H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects EUH032 Contact with acids liberates very toxic gas P280 Wear protective gloves protective clothing eye protection face protection P305 P351 P338 IF IN EYES Rin
16. in down the sample and 15 50 ml urine fresh frozen resuspend the pellet in 100 ul ddH 0 5 10 mg tissue sample fresh frozen homogenize sample 5 10 mg biopsy fresh frozen completely by crushing and fill up to 100 ul with ddH2O or 5 10 mg frozen section fresh frozen 1x PBS Buffer fill up to 100 ul with buffer 0 4 SDS 50mM Tris HCl swabs fresh dried pH 8 0 or use 120 ul of transport medium or rinse liquid 10 100 ul bacterial culture fresh frozen fill up to 100 ul with ddH O cell pellet from 1x 10 bacteria fresh frozen resuspend in 100 ul ddH20 Table 1 9 Sample treatment before starting also see table 1 Blood buffy coat saliva synovial fluid see table 1 Urine Spin down up to 50 ml and discard the supernatant suspend the cells in ddH 0 Tissue Homogenize or crush the tissue sample and fill it in a 2 ml 96 Deep Well Plate Swab Can be provided in transportation medium take 120 ul of this media an mix it with 80 ul ddH20 An untreated swab fresh or dried must be mixed with special buffer see table and the supernatant is filled in the well the yield can be increased if the swab remains in the well during the lysis 15 Invisorb Universal Bacterial HTS 96 Kit C 0413 Preparing reagents and buffers Prior to each isolation Before starting a run bring all reagents to room temperature Where necessary gently mix and re dissolve any precipitates by warming
17. ing steps Attention Please be aware that you have to prepare the Binding Buffer HL see page 16 Sample Preparation Homogenize the 5 10 mg biopsy samples in a Mixer Mill or under liquid nitrogen with mortar and pestle Normalization Add 100ul water or 1x PBS to each homogenate and resuspend the homogenate by pipetting up and down Transfer the resuspended sample into one well of the 2 ml Collection Plate and proceed the purification process Follow the Protocol 1 from step 2 The lysis step with Proteinase K may be extended to 15 min The lysis step with Lysis Buffer HL may be extended to 10 min 22 Invisorb Universal Bacterial HTS 96 Kit C 0413 Troubleshooting material Problem Probable cause Comments and suggestions clogged DNA viscous lysates too reduce amount of starting material or dilute the Binding Plate B much starting lysate in Lysis Buffer HL 1 1 and use the half before adding Binding Buffer HL if this happens during the run pierce well with a needle increase incubation time with Lysis Buffer HL low amounts of extracted DNA insufficient lysis insufficient binding of DNA onto the membrane incomplete elution low DNA concentration increase time for lysis with Lysis Buffer HL reduce amount of starting material mix the sample vigorously with Lysis Buffer HL before and during lysis by pipetting up and down or shaking incubate at 65 C after adding Lysis Buffer HL ina incubator
18. late 6 Add 600 ul Wash Buffer I to each well of the DNA Binding Plate B Cover the DNA Binding Plate B with the Plate Lid Load the whole block DNA Binding Plate B 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 3 min at RT Remove the cover 19 Invisorb Universal Bacterial HTS 96 Kit C 0413 and discard the filtrate and place the DNA Binding Plate B back to the top of the 2 ml Collection Plate Add 700 ul Wash Buffer II to each well of the DNA Binding Plate B Cover the DNA Binding Plate B with the Plate Lid Load the whole block DNA Binding Plate B 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket Centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 3 min at RT Remove the cover and empty the 2 ml Collection Plate and dry its upper side with paper Place the DNA Binding Plate B onto a clean surface paper towel Repeat step 7 Cover the DNA Binding Plate B with the Plate Lid and put it on top of the 2 ml Collection Plate Load the whole block DNA Binding Plate B 2 ml Collection Plate into the holder and place the whole assembly in the rotor bucket of the centrifuge Centrifuge at maximum speed for at least 10 min at RT to eliminate any traces of ethanol Take the DNA Binding Plate B 2 ml Collection Plate out of the centrifuge remove the cover and place the plate on a clean paper towel Discard
19. nt of Elution Buffer min 75 ul This may result in a higher DNA concentration Eluting twice with each with 75 ul Elution Buffer is recommended and produces lightly higher yield 3 Eluting with pre warmed Elution Buffer up to 80 C increases yield 4 It is recommended to centrifuge the isolated DNA for 1 min at maximum speed before starting any application 5 All centrifugations are hold at room temperature 6 To avoid cross contaminations plates have to be sealed with Sealing Foils 7 It is recommended to use multi channel pipettes esp electric multi channel pipettes with a capacity of 1 ml 8 Bacteria must be cultivated under special conditions and an aliquot of the bacteria suspension is used to win a bacteria pellet by centrifugation at high speed for 5 min The supernatant is removed Sample preparation For further information contact STRATEC Molecular Bacteria must be cultivated under special conditions and an aliquot of the bacteria suspension is used to get a bacteria pellet by centrifugation at high speed for 5 min The supernatant is removed 14 Invisorb Universal Bacterial HTS 96 Kit C 0413 General considerations A summary of sample setup is described in the table below Sample and sample size Condition Treatment fresh frozen with 10 100 ul blood anticoagulants fill up to 100 ul with ddH O 10 100 ul serum plasma __ fresh frozen fill up to 100 ul with ddH O sp
20. on media use 100 ul of this medium Transfer 100 ul of the rinsed liquid or of the transportation media of one swab into the well of the 2 ml Collection Plate and proceed the purification process Follow the Protocol 1 from step 2 21 Invisorb Universal Bacterial HTS 96 Kit C 0413 Protocol 5 Purification of bacterial DNA from urine samples using a centrifuge Please read the protocols prior to the start of the preparation and complete preparing steps Attention Please be aware that you have to prepare the Binding Buffer HL see page 16 Sample Preparation Centrifuge the collected urine sample 15 50 ml for 15 minutes at 1 500 x g 3500 rpm Decant the supernatant carefully and resuspend the sediment with 3 ml 1 x PBS Centrifuge for 5 minutes at 1 500 x g 3500 rpm Decant the supernatant carefully but completely by inverting the tube for some minutes It is important to remove the supernatant completely Residual amounts of liquid will have negative influence on the further extraction procedure Normalization Add 100 ul water or 1x PBS to each pellet and resuspend the pellet by pipetting up and down Transfer the resuspended sample into one well of the 2 ml Collection Plate and proceed the purification process Follow the Protocol 1 from step 2 Protocol 6 Purification of bacterial DNA from small biopsy samples using a centrifuge Please read the protocols prior to the start of the preparation and complete prepar
21. orb technology is covered by patents and patent applications US 6 110363 US 6 043 354 US 6 037 465 EP 0880535 WO 9728171 WO 9534569 EP 0765335 DE 19506887 DE 10041825 2 WO 0034463 Invisorb is a registered trademark of STRATEC Biomedical AG The PCR process is covered by US Patents 4 683 195 and 4 683 202 and foreign equivalents owned by Hoffmann La Roche AG 2013 STRATEC Molecular all rights reserved for automated use please contact in Germany 030 9489 2901 10 or your local distributor Invisorb Universal Bacterial HTS 96 Kit C 0413 Contents Kit contents of Invisorb Universal Bacteria HTS 96 Kit C 3 Symbol 4 Storage 4 Quality control 4 Intended use 5 Product use limitations 5 Safety information 6 Product characteristic of Invisorb Universal Bacteria HTS 96 Kit C 7 Principle and Procedure 12 Important notes 14 Important points before staring the protocol 14 Important indications 14 Preparing reagents and buffers 16 Equipment and reagents to be supplied by user 17 Handling options 17 Scheme of Invisorb Universal Bacteria HTS 96 Kit C 18 DNA Isolation from clinical specimen using a centrifuge Protocol 1 Purification of bacterial DNA from clinical samples 19 Protocol 2 Purification of bacterial DNA from bacterial cultures 21 Protocol 3 Purification of bacterial DNA from food samples 21 Protocol 4 Purification of bacterial DNA from swab 21 Protocol 5 Purification of bacterial DNA from urine 22
22. ors such host strain inoculation antibiotic and type of culture medium The bacteria will be pelleted after cultivation Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size 12 Invisorb Universal Bacterial HTS 96 Kit C 0413 Urine The bacteria must be pelleted and the supernatant completely removed urea contaminations can inhibit PCR reactions Best results are obtained with fresh peletted material or bacteria pellets that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size The amount of purified DNA from max 15 50 ml urine depends from the included bacteria titre Pathogens in food material Listeria ssp For the detection of bacteria Listeria in foods they must be enriched and cultivated following the EU regulations and 35 of the food law An aliquot of the culture will only be used and the bacteria will be pelleted after cultivation Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size Procedure Normalization Each sample is mixed with PBS or ddH20 and filled up
23. own below From this kit only the results for the S aureus detection are shown Now Fin cree r z Fig 5 Shown are the amplification curve for 2 S aureus positive samples red and green and the NTC black no template control Tab 5 Ct values for the detection of S aureus FAM and the internal amplification control JOE Colour Name Genotype Ct FAM Ct JOE HH taureus 50 S aureus 97 33 34 32 positive a 2mrsa50 S aureus 30 09 34 34 positive HH taureuss S aureus 32 19 34 47 positive E 2mrsa5 gt aureus 34 38 negative E PTC S aureus 3371 37 22 positive PTC S aureus 33 02 35 26 positive o NTC S aureus 30 60 negative NTC S aureus 30 62 negative Results Reproducibility of PCR values showed no evidence of PCR inhibitors in any of the extracted bacterial DNA samples Also dilutions and comparison between different extraction and matrices showed high reproducibility The usability of more clinical relevant bacteria was demonstrated with inactivated S aureus but the results are not directly comparable with real clinical samples 11 Invisorb Universal Bacterial HTS 96 Kit C 0413 Principle and procedure The Invisorb Universal Bacteria HTS 96 Kit procedure comprises following steps sample pre treatment and normalization of all samples to a volume of 100 ul lysis of bacterial cells binding the DNA to the membrane washing
24. performance of all components of the Invisorb Universal Bacteria HTS 96 Kit have been tested separately against predetermined specifications routinely on lot to lot to ensure consistent product quality If you have any questions or problems regarding any aspects of Invisorb Universal Bacteria HTS 96 Kit or other STRATEC Molecular products please do not hesitate to contact us A copy of STRATEC Molecular s terms and conditions can be obtained upon request or are presented at the STRATEC Molecular webpage For technical support or further information please contact from Germany 49 0 30 9489 2901 2910 from abroad 49 0 30 9489 2907 or contact your local distributor Invisorb Universal Bacterial HTS 96 Kit C 0413 Intended use The Invisorb Universal Bacteria HTS 96 Kit has been designed to extract highly pure DNA from up to 100 ul of fresh or frozen blood plasma serum cell free body fluids of human origin like urine as well as rinsed liquid from swabs or cell pellets in a 96 well format using a centrifuge Fresh or frozen blood plasma or serum from common blood collection systems can be used with EDTA or citrate but not with heparin Final extracted bacterial DNA is of high quality and suitable for a wide variety of downstream applications For reproducible and high yields appropriate sample storage is essential Yields may vary from sample to sample depending on factors such as the health of the donor patient medication o
25. plied nor express The user is responsible to validate the performance of the STRATEC Molecular Product for any particular use STRATEC Molecular does not provide for validation of performance characteristics of the Product with respect to specific applications STRATEC Molecular Products may be used e g in clinical diagnostic laboratory systems conditioned upon the complete diagnostic system of the laboratory the laboratory has been validated pursuant to CLIA 88 regulations in the U S or equivalents in other countries All Products sold by STRATEC Molecular are subject to extensive quality control procedures according to ISO 9001 2000 and ISO EN 13485 and are warranted to perform as described herein Any problems incidents or defects shall be reported to STRATEC Molecular immediately upon detection thereof The chemicals and the plastic parts are for laboratory use only they must be stored in the laboratory and must not be used for purposes other than intended The Product with its contents is unfit for consumption Invisorb Universal Bacterial HTS 96 Kit C 0413 Safety information When and while working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Avoid skin contact Adhere to the legal requirements for working with biological material For more information please consult the appropriate material safety data sheets MSDS These are available online in convenient and compact PDF
26. r more information please consult the appropriate material safety data sheets MSDS see our webpage www stratec com Measuring cylinder 250 ml Disposable gloves Multichannel pipette with tips Reagents reservoirs for multichannel pipettes 99 8 ethanol Isopropanol Centrifuge output 2 2 000 x g is necessary o Sigma Centrifuge 4 15C or centrifuge 4K15C with plate rotor 2 x 96 or other rotors o Eppendorf Centrifuge 5804 5804 R 5810 5810 R with Deepwell Plate Rotor A 2 DWP oo O00 0 0 0 The Invisorb Universal Bacteria HTS 96 Kit C is validated with 2 Propanol Rotipuran gt 99 7 p a ACS ISO Order no 6752 from Carl Roth Possible suppliers for lsopropanol Carl Roth 2 Propanol Applichem Sigma Rotipuran gt 99 7 p a ACS ISO 2 Propanol fur die Molekularbiologie 2 Propanol Order no 6752 Order no A3928 Order no 59304 1L F Possible supplier for centrifuges KNF Neuberger GmbH VACUUBRAND GMBH CO KG Alter Weg 3 Alfred Zippe Str 4 D 79112 Freiburg D 97877 Wertheim Phone 49 0 7664 5909 0 Phone 49 0 9342 808 0 Fax 49 0 7664 5909 99 Fax 49 0 9342 59880 E Mail info knf de E Mail info vacuubrand de 17 Invisorb Universal Bacterial HTS 96 Kit C 0413 Scheme of Invisorb Universal Bacteria HTS 96 Kit C Please read protocols prior to the start of the preparation Normalization each sample smaller than 100 ul must be mixed with ddH20 or PBS buffer filled up to a final
27. r sample storage conditions The purified DNA is of high quality and free of proteins nucleases and other impurities and is ready to use for different downstream applications like PCR quantitative PCR real time PCR or other routine diagnostic methods Any diagnostic results generated using the sample preparation procedure in conjunction with any downstream diagnostic assays should be interpreted with regard to other clinical o laboratory finding THE PRODUCT IS INDENTED FOR USE BY PROFESSIONAL USERS ONLY SUCH AS TECHNICIANS PHYSICIANS AND BIOLOGISTS TRAINED IN MOLECULAR BIOLOGICAL TECHNIQUES It is designed to be used with any downstream application employing enzymatic amplification or other enzymatic modifications of RNA followed by signal detection or amplification Any diagnostic results generated by using the sample preparation procedure in conjunction with any downstream diagnostic assay should be interpreted with regard to other clinical or laboratory findings To minimize irregularities in diagnostic results adequate controls for downstream applications should be used Product use limitation The kit is neither validated for the isolation of human or bacterial genomic DNA from faecal samples nor for viral nucleic acids or RNA isolation The included chemicals are only useable once Differing of starting material or flow trace may lead to inoperability therefore neither a warranty nor guarantee in this case will be given neither im
28. rial that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size Use of poor quality starting material also leads to reduced length and yield of purified DNA The amount of purified DNA in the Invisorb Universal Bacteria HTS 96 Kit C procedure from max 10 mg biopsy sample depends on the nature of starting material Body fluids plasma serum synovial fluids urine Best results are obtained with fresh material or material that has been immediately frozen and stored at 20 C or 80 C Repeated freezing and thawing of stored samples should be avoided since this leads to reduced DNA size The amount of purified DNA in the Invisorb Universal Bacteria HTS 96 Kit C procedure from max 100 ul body fluids e g amniotic fluid synovial fluid depends on the nature and amount of cells Swabs The protocol works with fresh prepared swabs as well as with dried swabs Please note that stored and dried swab sample often characterized by isolation of apoptotic DNA visible on agarose gel as typical apoptotic DNA banding pattern You can use also up to 100ul rinsed liquid from swab The protocol has not been validated for isolation of DNA from swabs which are stored under a storage buffer Bacterial cultures Bacterial cultures grow in the presence of a selective agent such as an antibiotic The yield and quality of DNA may depend on fact
29. s and in different matrices The cells were grown in an over night culture and cell pellets from 1ml of this culture is stored at 20 C until use For all experiment always a fresh pellet was taken from 20 C and the cells were discarded after the use for one day The detection was done by an in house Bacillus subtilis real time PCR based detection assay All real time PCR s were performed on a Corbett Rotor Gene 3000 7 Invisorb Universal Bacterial HTS 96 Kit C 0413 PCR Inhibitor and Cross Contamination Test To maximize the detection of any potential contamination event positive and no sample controls were arranged in alternating wells in a checkerboard pattern Fig 1 Out of those samples Fig 2 shows a real time PCR run of the extracted DNA PCR were done with an in house SYBR Green based real time PCR assay on a Corbett Rotor Gene 3000 machine N OOOOOOECO JO JS JOL IOR GL JO JO JO IS OBOO8OEO 0000000 OBOOOEO JOL JOL JO Ji SL JO JO JOE JO JO JO OBO8OEO Fig 1 Checkerboard Pattern utilized for the cross contamination analysis test Samples red and no sample controls white arranged in alternating wells Fig 2 Real time PCR results from positive samples red and no sample controls green arranged in a Checkerboard NTC no template control black and PTC positive template control pink are also shown Tab 1 To show the high reproducibility of the Ct
30. se cautiously with water for several minutes Remove contact lenses if present and easy to do Continue rinsing P310 Immediately call a POISON CENTER or doctor physician P405 Store locked up P273 Avoid release to the environment Emergency medical information can be obtained 24 hours a day from infotrac outside of USA 1 352 323 3500 in USA 1 800 535 5053 6 Invisorb Universal Bacterial HTS 96 Kit C 0413 Product characteristic of the Invisorb Universal Bacteria HTS 96 Kit Starting material Yield Time for preparation Ratio up to 100 ul whole blood up depending on the amount about 80 min A260 A 280 5 10 mg tissue and kind of starting 1 7 1 9 up to 100 ul cell free body material fluids serum plasma synovial liquid urine swabs The Invisorb Universal Bacteria HTS Kit is designed for isolation and purification of bacterial DNA from bacterial pellets or different clinical samples e g up to 100 ul of fresh or frozen blood plasma serum cell free body fluids of human origin like urine as well as rinsed liquid from swabs or cell pellets on a 96 well format The kit composition of the Invisorb Universal Bacteria HTS Kit C is designed for use on a centrifuge In a manual step all samples are normalized to a sample volume of 100 ul before using Small samples should be adjusted to 100 ul with 1x PBS or water before starting the protocol The kit is designed for
31. shearing of genomic DNA is to use special tips that have wide openings designed for pipetting genomic DNA 4 DNA Yield The amount of purified DNA from the whole blood depends on the leucocytes content sample source transport storage and age Various different primary tubes and anticoagulants except heparin can be used to collect blood samples for the Invisorb procedure 24 Invisorb Universal Bacterial HTS 96 Kit C 0413 Ordering information Product Invisorb Universal Bacteria HTS 96 Kit C Invisorb Universal Bacteria HTS 96 Kit C Invisorb Universal Bacteria HTS 96 Kit C InviMag Universal Bacteria Kit KF96 InviMag Universal Bacteria Kit KF96 InviMag DNA Bacteria Mini Kit KFmL InviMag DNA Bacteria Mini Kit KFmL RTP Bacteria DNA Mini Kit RTP Bacteria DNA Mini Kit RTP Mycobacteria Kit RTP Mycobacteria Kit Lysozyme Lysozyme Buffer 25 Catalogue No 7033300200 7033300300 7033300400 7433300100 7433300100 2433150100 2433150200 1033200200 1033200300 1033220200 1033220300 3020401300 3020401400 Package Size 2 x 96 preps 4 x 96 preps 24 x 96 preps 1 x 96 preps 5 x 96 preps 15 preps 75 preps 50 preps 250 preps 50 preps 250 preps 150 mg 15 ml Invisorb Universal Bacterial HTS 96 Kit C 0413 stratecee molecular STRATEC Molecular GmbH Robert Rossle Str 10 13125 Berlin Germany Phone 49 30 94 89 29 01 Fax 49 30 94 89 29 09
32. simultaneous processing of multiple samples The samples are pretreated with Lysozyme at 37 C to break the bacterial cell wall and lysed in an optimized lysis buffer Proteins are degraded during the lysis with Proteinase K at 65 C The lysis efficiency is improved by shaking the samples in the 96 well plate during the lysis The DNA binds to a filter membrane followed by washing steps and the final elution The procedure requires minimal interaction by the user allowing safe handling of potentially infectious samples The procedures are designed to avoid sample to sample cross contamination The Invisorb Universal Bacteria HTS 96 Kit yield highest quality of DNA so the obtained DNA can be used directly in o PCR o Realtime PCR o RFLP analysis o Restriction Enzyme Digestion Proven Invisorb 96 technology provides uniform DNA recovery and purity across the 96 well plate with no cross contamination between samples Protocol Validation Verification Testing Bacterial DNA extraction protocol was functionally tested on a centrifuge using Invisorb Universal Bacteria HTS 96 Kit C provided reagents and consumables Typical results for the extraction of bacterial DNA from buffer plasma and blood are shown below Actual results will vary depending upon sample age quality type and species of subject Samples For testing frozen cell pellets of the gram positive bacterium Bacillus subtilis were used in different dilutions of cell
33. spect the Product and its components as well as the package for any apparent damages correct quantities and quality If there are any unconformities you have to notify STRATEC Molecular in writing with immediate effect upon inspection thereof If buffer bottles are damaged contact the STRATEC Molecular Technical Services or your local distributor In case of liquid spillage refer to Safety Information see page 7 Do not use damaged kit components since their use may lead to poor kit performance o Always change pipet tips between liquid transfers To avoid cross contamination we recommend the use of aerosol barrier pipet tips o All centrifugation steps are carried out at room temperature When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles Discard gloves if they become contaminated Do not combine components of different kits unless the lot numbers are identical Avoid microbial contamination of the kit reagents To minimize the risk of infections from potentially infectious material we recommend working under laminar air flow until the samples are lysed o This kit should only be used by trained personnel Q O O7 OO Important indications 1 Optimal disruption of tissue is important for obtaining maximum yield and purity of genomic DNA For high sample throughput it is recommended to use a Mixer Mill with a 96 insert 2 The elution can be done by using lower amou
34. to 30 C until dissolved Swirl gently to avoid foaming Lysis Buffer HL and Elution Buffer are ready to use Proteinase K Add described amount of ddH20 see table below to needed tube or bottle of Proteinase K mix thoroughly and store not needed Proteinase K at 20 C Dividing the Proteinase K into aliquots to avoid repeated freezing and thawing is recommended Lysozyme Lysozyme should be prepared directly before starting the isolation Solve the lyophilized Lysozyme within the bottle of Lysozyme Buffer Mix thoroughly until all of the Lysozyme is solved Store not directly used Lysozyme solution in aliquots at 20 C and avoid repeated freezing and thawing Wash Buffer I and Wash Buffer II Before use add the described volume of 96 100 ethanol to the bottle with Wash Buffer and Il as described below After adding the ethanol mix shortly and keep the bottles always firmly closed 2 x 96 DNA extractions Add 24 ml 99 7 Isopropanol to each Binding Buffer HL Mix by intensive shaking by inverting for 1 min Shortly before use mix by inverting several times Add 80 ml 96 100 ethanol to the bottle Wash Buffer I Add 105 ml 96 100 ethanol to the bottle Wash Buffer II Add 1 5 ml ddH2O to the Proteinase K Mix thoroughly and store at 20 C Add provided amount of Lysozyme to the bottle with the Lysozyme Buffer and mix it thoroughly Aliquot and store at 20 C 100 ul Lysozyme mixture sample 4x 96 DNA extractions
35. to a final volume of 100 ul Sample preparation Bacteria must be cultivated under special conditions and an aliquot of the bacteria suspension will be used to get a bacteria pellet by centrifugation at high speed for 5 min The supernatant will be removed Lysis Lysis is performed in several steps at first in the presence of Lysozyme to break the cell wall of the bacteria Then samples are lysed under denaturing conditions at different elevated temperatures and continuously shaking using a Lysis Buffer and Proteinase K to digest the proteins DNases are inactivated The bacterial DNA is secured Unlysed sample parts should be removed before the binding step Binding genomic DNA By adding Binding Buffers HL to the lysate optimal binding conditions are achieved Each lysate is then applied to a well of the DNA Binding Plate B and genomic DNA is adsorbed onto the membrane Removing residual contaminants Contaminants are efficiently washed away using Wash Buffers while the bacterial genomic DNA remains bound to the membrane Elution of pure genomic DNA Genomic DNA is eluted from the Invisorb 96 Filter plate using 100 ul Elution Buffer The eluted DNA is ready for use in different downstream applications Long term studies on the stability of eluates are still in progress 13 Invisorb Universal Bacterial HTS 96 Kit C 0413 Important notes Important points before starting a protocol Immediately upon receipt of the Product in
36. tory Press Cold Spring Harbor NY 23 Invisorb Universal Bacterial HTS 96 Kit C 0413 Appendix General notes on handling DNA 1 Nature of DNA The length and physical nature of DNA requires careful handling to avoid damage due to shearing and enzymatic degradation Other conditions that affect the integrity and stability of DNA include acidic and alkaline environments high temperature and UV irradiation Careful isolation and handling of high molecular weight DNA is necessary to ensure compatibility with various downstream applications Damaged DNA could perform poorly in applications such as genomic Southern blotting long template PCR 2 Storage of DNA A working stock of DNA can be stored at 2 4 C for several weeks For long term storage DNA should be stored at 20 C but storing at 20 C can cause shearing particularly if the DNA is exposed to repeated freeze thaw cycles Note that the solution in which the nucleic acid is eluted in will affect it s stability during storage Pure water lacks buffering capacity and an acidic pH may lead to acid hydrolysis Tris or Tris EDTA buffer contains sufficient buffering capacity to prevent acid hydrolysis 3 Drying dissolving and pipetting DNA Avoid overdrying genomic DNA after ethanol precipitation It is better to let it air dry than to use a vacuum Avoid vigorous pipetting Pipetting genomic DNA through small tip openings causes shearing or nicking One way to decrease
37. volume of 100 ul and transferred in a2 ml Collection Plate Add 100 ul diluted Lysozyme to each sample Incubate at RT for 10 min Add 20 ul Proteinase K to each sample Mix completely Incubate at 65 C for 10 min 800 rpm Add 200 ul Lysis Buffer HL to each well and mix completely Incubate at 65 C under continuously shaking for 3 min 800 rpm Add 200 ul Binding Buffer HL follow preparing instructions to each well mix completely place the DNA Binding Plate B on the top of a 2 ml Collection Plate transfer lysates completely to the DNA Binding Plate B incubate at RT for 1 min centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 6 min at RT or until all lysate was running trough discard filtrate and place the DNA Binding Plate B back on the top of a 2 ml Collection Plate add 600 ul Wash Buffer I to each well of DNA Binding Plate B centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 3 min at RT discard filtrate and place the DNA Binding Plate B back on the top of a 2 ml Collection Plate add 700 ul Wash Buffer II to each well of DNA Binding Plate B centrifuge at 1 400 1 700 x g 3 700 4 000 rpm for 3 min at RT discard filtrate and place the DNA Binding Plate B back on the top of a 2 ml Collection Plate Repeat the Wash Buffer Il step remove all waste from the Collection Plate centrifuge at maximum
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