Genomic DNA from Forensic Samples
Contents
1. to lysis buffer FLB quality and or suboptimal Carryover of ethanol performance of genomic e Be certain to centrifuge gt 5 min at 3 000 x g in order to remove DNA in all of ethanolic buffer B5 before eluting the DNA If for any enzymatic reason the level of buffer B5 has reached the column outlet reactions after the second wash discard flow through Place the NucleoSpin DNA Trace F column back into the collecting tube and centrifuge again MACHEREY NAGEL 06 2006 Rev 04 15 Genomic DNA from Forensic Samples 6 2 Ordering information Product Cat No Pack of NucleoSpin DNA Trace kit 740942 4 4 preps NucleoSpin DNA Trace kit 740942 25 25 preps NucleoSpin funnel column 740959 30 columns Wash buffer BW 740922 100 ml aed B5 concentrate 740921 20 ml Pupa p DNA Trace bones 74094325 PI Proteinase K 740506 100 mg RNase A 740505 100 mg RNase A 740505 50 50 mg 16 MACHEREY NAGEL 06 2006 Rev 04 Genomic DNA from Forensic Samples 6 3 Product use restriction warranty NucleoSpin DNA Trace kit components were developed designed and sold for research purposes only They are suitable for in vitro uses only No claim or representation is intended for its use to identify any specific organism or for clinical use diagnostic prognostic therapeutic or blood banking It is rather the responsibility of the user to verify the use of the NucleoSpin DNA Trace kit for a specific application range as the performance2. characteristic of this kit has not been verified to a specific organism This MACHEREY NAGEL product is shipped with documentation stating specifications and other technical information MACHEREY NAGEL warrants to meet the stated specifications MACHEREY NAGEL s sole obligation and the customer s sole remedy is limited to replacement of products free of charge in the event products fail to perform as warranted Supplementary reference is made to the general business terms and conditions of MACHEREY NAGEL which are printed on the price list Please contact us if you wish an extra copy MACHEREY NAGEL does not warrant against damages or defects arising in shipping and handling transport insurance for customers excluded or out of accident or improper or abnormal use of this product against defects in products or components not manufactured by MACHEREY NAGEL or against damages resulting from such non MACHEREY NAGEL components or products MACHEREY NAGEL makes no other warranty of any kind whatsoever and SPECIFICALLY DISCLAIMS AND EXCLUDES ALL OTHER WARRANTIES OF ANY KIND OR NATURE WHATSOEVER DIRECTLY OR INDIRECTLY EXPRESS OR IMPLIED INCLUDING WITHOUT LIMITATION AS TO THE SUITABILITY REPRODUCTIVITY DURABILITY FITNESS FOR A PARTICULAR PURPOSE OR USE MERCHANTABILITY CONDITION OR ANY OTHER MATTER WITH RESPECT TO MACHEREY NAGEL PRODUCTS In no event shall MACHEREY NAGEL be liable for claims for any other damages whether direct indir
3. nent Contents Symbol Phrases Phrases guanidine x Xn Flammable R 10 22 S 7 16 25 BW hydrochloride Harmful if swallowed 36 38 isopropanol lt 25 Irritating to eyes and skin Proteinase K x Xn Irritating to eyes respiratory R S 22 24 Proteinase iyophilized Xi system and skin may cause 36 37 38 26 36 37 K sensitization by inhalation 42 guanidine Substance does not have to be specially labeled as hazardous FLB hydrochloride lt 10 Risk Phrases R 10 R 16 R 22 R 36 37 38 R 36 38 R 42 Flammable Keep away Harmful if swallowed Irritating to eyes respiratory system and skin Irritating to eyes and skin May cause sensitization by inhalation Safety Phrases S7 S16 S 22 S 24 S25 S 26 S 36 37 Keep container tightly closed Keep away from sources of ignition No Smoking Do not breathe dust Avoid contact with the skin Avoid contact with the eyes In case of contact with eyes rinse immediately with plenty of water and seek medical advice Wear suitable protective clothing and gloves Label not necessary if quantity below 125 g or ml concerning 67 548 EEC Art 25 1999 45 EC Art 12 and German GefStoffV 42 and TRGS 200 7 1 8 MACHEREY NAGEL 06 2006 Rev 04 NucleoSpin DNA Trace 5 Standard protocol for the isolation of genomic DNA from solid samples e g small amounts of cells or tissue forensic samples 1 Lyse sample Place the sample in a 15 ml centrifuge t
4. Genomic DNA from Forensic Samples User manual NucleoSpin DNA Trace June 2006 Rev 04 MACHEREY NAGEL MN Protocol at a glance Rev 04 Genomic DNA from Forensic Samples Funnel NucleoSpin DNA Trace 1 Lyse sample 4 8 ml FLB 50 ul proteinase K 56 C 1h 2 Clarify sample 10 min 5 000 x g 3 Adjust DNA binding conditions 3 5 ml ethanol vortex 4 Bind DNA Sant Load sample 3 min 3 000 xg 5 Washsilica Gut 4 wash 2 5 ml BW membrane 2 wash 5 ml B5 3 wash 5 ml B5 1 2 and 3 wash mn 3 000 xg 6 Dry silica membrane 10 min 3 000 xg 7 Elute highly pure DNA Sot 100 pl BE 70 C RT 2 min 3 min 3 000 x g MACHEREY NAGEL GmbH amp Co KG Neumann Neander Str 6 8 D 52355 D ren Germany Tel 49 0 24 21 969 270 Fax 49 0 24 21 969 279 e mail tech bio mn net com Genomic DNA from Forensic Samples Table of contents 1 Kit contents 4 2 Product description 5 2 1 The basic principle 5 2 2 About this user manual 5 2 3 Kit specifications 5 3 Storage conditions and preparation of working solutions 7 4 Safety instructions risk and safety phrases 8 5 Standard protocol for the isolation of genomic DNA from solid samples e g small amounts of cells or tissue forensic samples 9 5 1 Support protocol for the isolation of genomic DNA from human bones 12 6 Appendix 15 6 1 Troubleshooting 15 6 2 Ordering information 16 6 3 Product use res
5. ect incidental compensatory foreseeable consequential or special including but not limited to loss of use revenue or profit whether based upon warranty contract tort including negligence or strict liability arising in connection with the sale or the failure of MACHEREY NAGEL products to perform in accordance with the stated specifications This warranty is exclusive and MACHEREY NAGEL makes no other warranty expressed or implied The warranty provided herein and the data specifications and descriptions of this MACHEREY NAGEL product appearing in MACHEREY NAGEL published catalogues and product literature are MACHEREY NAGEL s sole representations concerning the product and warranty No other statements or representations written or oral by MACHEREY NAGEL s employees agent or representatives except written statements signed by a duly authorized officer of MACHEREY NAGEL are authorized they should not be relied upon by the customer and are not a part of the contract of sale or of this warranty MACHEREY NAGEL 06 2006 Rev 04 17 Genomic DNA from Forensic Samples Product claims are subject to change Therefore please contact our Technical Service Team for the most up to date information on MACHEREY NAGEL products You may also contact your local distributor for general scientific information Applications mentioned in MACHEREY NAGEL literature are provided for informational purposes only MACHEREY NAGEL does not warrant that al
6. iagnostics MACHEREY NAGEL 06 2006 Rev 04 5 Genomic DNA from Forensic Samples especially forensic samples may contain only traces of DNA However the amount will be sufficient for amplification and detection reactions e Additional enzymes which are not included in the kit may be necessary for lysis of certain bacteria e g lysozyme lysostaphine e Support protocol for the isolation of genomic DNA from human bones For this application additional buffers T1 B3 and proteinase K are necessary Therefore MACHEREY NAGEL has combined the NucleoSpin DNA Trace bones buffer set see ordering information This buffer set is especially designed for completion of the NucleoSpin DNA Trace kit It is suited for 25 preparations of genomic DNA from human bones in conjunction with the NucleoSpin DNA Trace kit Cat No 740942 25 Kit specifications at a glance DNA Trace Funnel Forensic samples which can be extracted with up to 8 ml lysis Sampl PERAS buffer FLB in general 10 mg tissue lt 10 cells Typical gt 70 for amounts gt 10 ng Recovery rate Typical traces of DNA at least 1 ng Sensitivity Elution volume 100 ul Purity ready to use for subsequent amplification reactions Binding capacity 20 ug DNA Time prep 60 min without proteinase K incubation which needs gt 1h Column type NucleoSpin DNA Trace F columns fitting in 50 ml tubes 6 MACHEREY NAGEL 06 2006 Rev 04 Genomic DNA fro
7. l applications have been tested in MACHEREY NAGEL laboratories using MACHEREY NAGEL products MACHEREY NAGEL does not warrant the correctness of any of those applications Please contact MACHEREY NAGEL Germany Tel 49 0 24 21 969 270 e mail TECH BlIO mn net com 18 MACHEREY NAGEL 06 2006 Rev 04
8. m Forensic Samples 3 Storage conditions and preparation of working solutions Attention Buffers FLB and BW contain guanidine hydrochloride Wear gloves and goggles e All kit components can be stored at room temperature 20 25 C and are stable for up to one year Before starting any NucleoSpin DNA Trace protocol prepare the following e Before first use of the kit add indicated volume of Proteinase Buffer to dissolve lyophilized proteinase K Proteinase K solution is stable at 4 C for up to 6 months Storage at 20 C is recommended if the solution will not be used up during this period NucleoSpin DNA Trace 4 preps 740942 4 25 preps 740942 25 Proteinase K 6 mg 30 mg add 300 ul add 1500 ul Proteinase Buffer Proteinase Buffer Buffer B5 20 ml 80 ml concentrate add 80 ml ethanol add 320 ml ethanol e Add indicated volume of 96 100 ethanol to the buffer B5 concentrate e Upon storage especially at low temperatures a white precipitate may form in buffer FLB Such precipitates have to be dissolved by incubating at 45 50 C for 10 min before use MACHEREY NAGEL 06 2006 Rev 04 7 Genomic DNA from Forensic Samples 4 Safety instructions risk and safety phrases The following components of the NucleoSpin DNA Trace kits contain hazardous contents Wear gloves and goggles and follow the safety instructions given in this section Compo ___ Hazard BEZK Risk Safety
9. mple Pipette mixture onto the NucleoSpin DNA Trace F column Centrifuge for 3 min at 3 000 x g Discard flow through C gt 3 min 3 000 xg 5 Wash silica membrane 1 wash 2 5 ml BW Add 2 5 ml buffer BW to the NucleoSpin DNA Trace F it 3 min column Centrifuge for 3 min at 3 000 x g SF 3 000 xg 2 wash L 5ml B5 Add 5 ml buffer B5 to the NucleoSpin DNA Trace F 3 min column Centrifuge for 3 min at 3 000 x g and discard 3 000 xg flow through 3 wash D Add 5 ml buffer B5 to the NucleoSpin DNA Trace F 3 min column Centrifuge for 3 min at 3 000 x g and discard 3 000 xg flow through i 5ml B5 6 Dry silica membrane 10 min Centrifuge additional 10 min at 3 000 x g in order to 3 000 x g remove buffer B5 completely 10 MACHEREY NAGEL 06 2006 Rev 04 NucleoSpin DNA Trace 7 Elute highly pure DNA Attach the supplied elution tube with adaptor to the 100 ul BE NucleoSpin DNA Trace F column and insert assembly im into a new 50 ml collection tube not provided Pipette ar RT 100 pl elution buffer BE preheated to 70 C onto the NucleoSpin membrane and incubate for 2 min at room Re 2min temperature S Centrifuge for 3 min at 3 000 x g to collect the nucleic i acid containing fraction eS 3 min 3 000 x g Remove the elution tube containing the nucleic acids and keep it for further use MACHEREY NAGEL 06 2006 Rev 04 11 NucleoSpin DNA Trace 5 1 Suppor
10. nol is removed during this step MACHEREY NAGEL 06 2006 Rev 04 13 NucleoSpin DNA Trace Elute highly pure DNA Attach the supplied elution tube with adaptor to the NucleoSpin DNA Trace F column and insert assembly into a new 50 ml collection tube not provided Add 60 ul elution buffer BE preheated to 70 C Incubate at room temperature for 2 min Centrifuge for 3 min at 3 000 x g to collect the nucleic acid containing fraction Remove the elution tube containing the nucleic acids and keep it for further use MACHEREY NAGEL 06 2006 Rev 04 Genomic DNA from Forensic Samples 6 Appendix 6 1 Troubleshooting Problem Possible cause and suggestions Incomplete lysis of sample e Sample not thoroughly homogenized and mixed with buffer FLB proteinase K The mixture has to be shaken continuously Alternatively prolong incubation time with proteinase K No or poor Reagents not applied properly DNA yield e Prepare buffer B5 and proteinase K solutions according to poor DNA instructions section 3 Add ethanol to lysates before loading quality them on NucleoSpin DNA Trace F columns Suboptimal elution of DNA from the column e Apply buffer BE 70 C directly onto the center of the silica membrane and incubate for 2 min Elution efficiencies decrease dramatically if elution is done with other buffers at pH lt 7 0 RNA in sample If RNA free DNA is desired add 20 ul of RNase A solution Poor DNA 20 mg ml
11. solation kit may refer to the Protocol at a glance instead of this User Manual The Protocol at a glance is designed to be used only as a supplemental tool for quick referencing while performing the purification procedure First time users are strongly advised to read this User Manual 2 3 Kit specifications e NucleoSpin DNA Trace kit is designed for the preparation of highly pure genomic DNA from small amounts of any tissue cells and forensic samples e g dried blood spots The NucleoSpin DNA Trace F columns included in the kit are ideally suited for collecting small amounts of nucleic acids from large volumes because these columns are shaped like a funnel combining a large volume capacity with a small diameter of the binding membrane F means funnel The DNA isolated by NucleoSpin DNA Trace F columns can be used directly for PCR or other enzymatic reactions e Age storage conditions quantity and consistency of samples can affect DNA quality and therefore the protocol may be adapted accordingly e g increasing incubation time For successful DNA preparation it is essential that the sample is lysed well and separated afterwards only clear lysates should be loaded onto NucleoSpin DNA Trace F columns in order to avoid clogging of the silica membrane e The NucleoSpin DNA Trace kit allows purification of up to 20 ug of pure genomic DNA with an A260 280 ratio of between 1 70 and 1 90 Some samples PCR is patented by Roche D
12. t protocol for the isolation of genomic DNA from human bones Before starting with the preparation please read remarks below Before starting with the preparation set incubators or water baths to 56 C and 70 C respectively Before elution equilibrate elution buffer BE to 70 C Attention The list numbers in this support protocol do not correspond with the list numbers in section 4 and protocol at a glance Additional buffer T1 B3 and proteinase K is necessary The NucleoSpin DNA Trace bones buffer set Cat No 740943 25 is especially designed for completion of the NucleoSpin DNA Trace kit It is suited for 25 preparations of genomic DNA from human bones in conjunction with the NucleoSpin DNA Trace kit Cat No 740942 25 Preparation of buffer B3 Transfer the total contents of buffer B1 to reagent B2 Mix well The resulting buffer B3 is stable for at least 5 months at room temperature in the dark For each prep 2 ml additional buffer has to be prepared 0 56 M EDTA 0 25 M PO pH 8 not included in the NucleoSpin DNA Trace bones buffer set 1 Prepare sample Mill 1 g bone to a fine powder 2 Pre Lysis Add 2 ml buffer 0 5M EDTA 0 25 M PO pH 8 and 7 ml buffer T1 and 100 ul proteinase K solution Vortex to mix Be sure that the samples are completely covered with lysis solution If processing several samples proteinase K and buffer T1 may be premixed directly before use Do never mix bufer T1 and pro
13. teinase K more than 10 15 min before addition to the sample proteinase K tends to self digestion in buffer T1 without substrate Incubate at 56 C overnight Afterwards incubate sample for 48h at 4 C on a shaking incubator MACHEREY NAGEL 06 2006 Rev 04 NucleoSpin DNA Trace 3 Lysis Vortex the samples Add 8 ml buffer B3 vortex vigorously and incubate at 70 C for 10 min Vortex briefly Centrifuge for 10 min at 5 000 x g and transfer the supernatant to a new microcentrifuge tube 4 Adjust DNA binding conditions Add 8 4 ml ethanol 96 100 to the sample and vortex vigorously 5 Bind DNA For each sample place one NucleoSpin DNA Trace F column into a 50 ml collecting tube Apply the sample successively to the column Centrifuge for 3 min at 3 000 x g Discard the flow through and place the column back into the collecting tube 6 Wash silica membrane 15t wash Add 3 ml buffer BW Centrifuge for 3 min at 3 000 x g Discard the flow through and place the column back into the collecting tube 2 wash Add 3 ml buffer B5 to the column and centrifuge for 3 min at 3 000 x g Discard the flow through and place the column back into the collecting tube 3 wash Add 3 ml buffer B5 to the column and centrifuge for 3 min at 3 000 x g Discard the flow through and place the column back into the collecting tube 7 Dry silica membrane Centrifuge the column for 10 min at 3 000 x g Residual etha
14. triction warranty 17 MACHEREY NAGEL 06 2006 Rev 04 3 Genomic DNA from Forensic Samples 1 Kit contents NucleoSpin DNA Trace 4 preps 740942 4 25 preps 740942 25 Buffer FLB 50 ml 250 ml ee ate 20 mi 80 mi Buffer BW 15 ml 75 ml Buffer BE 5 ml 15 ml Proteinase K 6 mg 30 mg Proteinase Buffer 0 8 ml 1 8 ml NucleoSpin DNA plus 60 mi collection i 25 tubes 0 5 ml elution tubes 4 25 50 ml collecting tubes 4 25 Protocol 1 1 For preparation of working solutions and storage conditions see section 3 4 MACHEREY NAGEL 06 2006 Rev 04 Genomic DNA from Forensic Samples 2 Product description 2 1 The basic principle With the NucleoSpin DNA Trace method DNA can be prepared from cells tissue and many other sources Lysis is achieved by incubation of homogenized samples in a solution containing chaotropic ions in the presence of proteinase K Appropriate conditions for binding of DNA to the silica membrane in the NucleoSpin DNA Trace F columns are created by chaotropic salt itself and added ethanol The binding process is reversible and specific to nucleic acids Contaminations are removed by repeated washing with 2 different ethanolic buffers Pure genomic DNA is finally eluted under low ionic strength conditions in a slightly alkaline elution buffer 2 2 About this user manual Experienced users who are performing the isolation of DNA from forensic samples using a NucleoSpin DNA Tace i
15. ube not included E and add 4 8 ml lysis buffer FLB The sample should be covered completely with lysis buffer FLB 4 8 ml FLB Solid samples should be homogenized by commercial tools pestle and mortar rotor stator homogeniser In general S 10 mg tissue lt 10 cells or any DNA containing solid sample can be used Forensic samples dried blood spots chewing gum swabs etc should be covered completely with lysis buffer Add 50 ul proteinase K stock solution mix by vortexing and incubate at 56 C in a shaking water bath fat until complete lysis is obtained 1 3 h or overnight Vortexing every 15 min 3 4 times leads to shorter lysis times if no shaking water bath incubator is available Final 56 C incubation at 70 C 100 C for 5 min may be recommended for 4h optimal denaturation and lysis of difficult samples e g dried old or clotted blood samples 2 Clarify sample 10 min Afterwards any insoluble particles remaining in the eS sample have to be removed by centrifugation for 10 min at 2 5 000 x g in order to avoid clogging of the NucleoSpin DNA Trace membrane 25 000 xg 3 Adjust DNA binding conditions Add 3 5 ml ethanol 96 100 to 4 ml cleared FLB 3 5 ml lysate and vortex the mixture Use proportionally up ethanol scaled volumes of ethanol if more FLB lysate has been prepared in step 1 vortex MACHEREY NAGEL 06 2006 Rev 04 9 NucleoSpin DNA Trace 4 Bind DNA im load sa
Download Pdf Manuals
Related Search