InviMag Forensic Kit / KingFisher mL
Contents
1. InviMag Forensic Kit KingFisher mL for extractions of genomic DNA from forensic samples using KingFisher mL instrument Thermo Electron Vers 1205 Kit components storage at room temperature Important Store the MAP Solution A at 4 C Store lyophilized Proteinase K at 2 8 C Store diluted Proteinase K at 20 C but repeated freezing and thawing will reduced the activity dramatically Dividing the Proteinase K into aliquots and storage at 20 C is recommended 15 extractions 75 extractions Lysis Buffer G 1x 10ml 1x50 ml 10 mg for 5 x 10 mg for Erpecinaseis 0 5 ml working solution 5 x 0 5 ml working solution MAP Solution A 1x 0 5 ml 2x1ml Binding Buffer T 1x8ml 1x30 ml Elution Buffer D 1x2ml 1x 15 ml 1x 18ml 1x 45 ml Vai Butter final volume 60 ml final volume 150 ml Receiver Tubes Tubes 1 5 ml 1x15 5x15 KingFisher ml Tip Combs Thermo Electron EAS Tae KingFisher ml Tube Strips Thermo Electron TAD pe Manual 1 1 Initial steps Add 42 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Dilute Proteinase K by addition of 0 5 ml of ddH O mix thoroughly and store like described below Add 105 ml of 96 100 ethanol to the bottle Wash Buffer mix thoroughly and keep the bottle always firmly closed Dilute Proteinase K by addition2. of 0 5 ml of ddH O mix thoroughly and store like described below Vers 1205 KingFisher software 2 6 2 KingFisher Software 2 6 2 is used to create protocols for the KingFisher KingFisher mL and KingFisher 96 instruments Once a protocol has been created the user can either transfer the protocol into the KingFisher instrument memory or run the protocol directly from the software Directly run protocols are not stored in the instrument memory Checking the PC requirements The table below lists the PC requirements for KingFisher Software 2 6 2 PC requirements Interface Serial communication port via an RS 232 full duplex interface Supported operating Microsoft Windows 2000 systems Microsoft Windows XP Professional Disk space 500 MB free disk space Processor Intel Pentium gt 700 MHz recommended Memory 220 MB RAM recommended Serial ports available 1 Pointing device Mouse or equivalent is necessary CD ROM drive 1 Monitor color SVGA monitor with at least 1024 x 768 settings resolution and at least a 16 bit color environment Service Packs installed Microsoft Windows 2000 Service Pack 4 or greater Microsoft Windows XP Professional Service Pack 2 or greater Browser Microsoft Internet Explorer 6 0 or greater installed If you do not have the correct Service Packs installed you can download them from the Microsoft web pages http www micro
3. of Lysis Buffer G and 25 ul of Proteinase K Vortex the tube for 5 s Incubate the sample at 56 C for at least 1 h under continuously shaking e g by using a thermomixer Incubation overnight is also possible We recommend the overnight lysis at 42 C under continously shaking After lysis time carefully transfer the lysed sample into the Tube A of the KingFisher tube strip avoid carry over of starting material optional spin down the starting material Add the 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use 4 Vers 1205 G Isolation of DNA from tissue samples Place approximately 0 5 20 mg of fresh or frozen tissue sample cut the material into small pieces into a 1 5 ml Reaction Tube tube Add 600 ul of Lysis Buffer G and 25 ul of Proteinase K Vortex the tube for 5 s Incubate the sample at 56 C for at least 1 h under continuously shaking e g by using a thermomixer Incubation overnight is also possible We recommend the overnight lysis at 42 C under continously shaking After lysis time carefully transfer the lysed sample into the Tube A of the KingFisher tube strip avoid carry over of starting material optional spin down the starting material Add the 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use H Isolation of DNA from nail clippings Place the nail clipp
4. transfer the DNA into a 1 5 ml reaction tube and centrifuge at maximum speed for 1 minute and pipet the DNA into a new tube 6 Vers 1205 InviMAG Forensic_mL protocol description 1 Sample lysate is incubated with magnetic particles for 3 minutes in tube A 2 Particles are washed with Wash Buffer for 50 sec in tube B 3 Particles are washed with Wash Buffer for 50 sec in tube C 4 A drying step 8 min is performed after washing step in tube C 5 DNA is released into Elution Buffer D in tube D 6 Particles are discarded into tube C Ordering Information KingFisher mL and consumables 5400050 KingFisher mL Magnetic Particle Processor 100 240 V 50 60 Hz 97002111 KingFisher mL tip comb 800 pcs 97002121 KingFisher mL tube 900 pcs 20x45 pcs 97002131 KingFisher mL Combi 60 tubes and tip combs for 60 samples 97002141 KingFisher mL Combi 240 tubes and tip combs for 240 samples 7 Vers 1205
5. C for at least 1 h under continuously shaking e g by using a thermomixer Incubation overnight is also possible We recommend the overnight lysis at 42 C under continously shaking After lysis time carefully transfer the lysed sample into the Tube A of the KingFisher tube strip avoid carry over of starting material optional spin down the starting material Add the 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use C Isolation of DNA from hair roots Place a single hair root or more into a 1 5 ml Reaction Tube Add 600 ul of Lysis Buffer G and 25 ul of Proteinase K and 30u1 1 M DTT not provided Vortex the tube for 5 s Incubate the sample at 56 C for at least 1 h under continuously shaking e g by using a thermomixer Incubation overnight is also possible We recommend the overnight lysis at 42 C under continously shaking After lysis time carefully transfer the lysed sample into the Tube A of the KingFisher tube strip avoid carry over of starting material optional spin down the starting material Add the 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use 3 Vers 1205 D Isolation of DNA from cigarette butts Remove of a small piece 3 5 mm of the brown filter paper or of a part of the filter and place the material in a 1 5 ml Reaction Tube Add 600 ul of Lysis Buf
6. fer G and 25 ul of Proteinase K Vortex the tube for 5 s Incubate the sample at 56 C for at least 1 h under continuously shaking e g by using a thermomixer Incubation overnight is also possible We recommend the overnight lysis at 42 C under continously shaking After lysis time carefully transfer the lysed sample into the Tube A of the KingFisher tube strip avoid carry over of starting material optional spin down the starting material Add the 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use E Isolation of DNA from bubble gum Cut a part of the bubble gum into small pieces and place the material into a 1 5 Reaction Tube Add 600 ul of Lysis Buffer G and 25 ul of Proteinase K Vortex the tube for 5 s Incubate the sample at 56 C for at least 3 h under continuously shaking e g by using a thermomixer Incubation overnight is also possible We recommend the overnight lysis at 42 C under continously shaking After lysis time carefully transfer the lysed sample into the Tube A of the KingFisher tube strip avoid carry over of starting material optional spin down the starting material Add the 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use F Isolation of DNA from stamps and envelopes Cut the material into small pieces and transfer it into a 1 5 ml Reaction Tube Add 600 ul
7. ings into a 1 5 ml Reaction Tube Add 600 ul of Lysis Buffer G and 25 ul of Proteinase K and 30 ul 1 M DTT not provided Vortex the tube for 5 s Incubate the sample at 56 C for at least 1 h under continuously shaking e g by using a thermomixer Incubation overnight is also possible We recommend the overnight lysis at 42 C under continously shaking After lysis time carefully transfer the lysed sample into the Tube A of the KingFisher tube strip avoid carry over of starting material optional spin down the starting material Add the 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use Important Notes 1 Using the kit for other kinds of forensic sample the selection of one of the described protocols is recommended 2 After lysis the automatic extraction on the KingFisher ml is identically for all types of starting materials So it is possible to extract the DNA from different sample types simultaneously 3 Optional for isolation of DNA from forensic samples containing extrem low amounts of DNA it could be helpful to add Carrier RNA to the Binding Step after lysis We recommend to use Carrier RNA e g Poly A RNA Roche Diagnostics No 108626 Dissolve the RNA in RNase free water to obtain a solution of 1 ug ul Divide aliquots and store at 20 C Do not freeze and thaw the aliquots for more than 3 times We recommend adding of 1 ul Carrier RNA
8. per sample 5 Vers 1205 2 KingFisher mL process Important Before starting the purification process with the KingFisher mL please read carefully the King Fisher mL user manual After finishing the sample lysis fill the tubes of the KingFisher mL strip tubes with the following Buffers respectively Please avoid evaporation of the prefilled buffer components by sealing the KingFisher tube strips with a sealing foil or with parafilm A Place an appropriate number of tube strips needed for the samples one tube strip per sample into removable tube strip tray Note Resuspend the magnetic particles MAP solution A thoroughly before use B Tube A Add 625 ul lysed sample 300 ul Binding buffer T and 20 ul MAP solution A C Tube B Add 800 ul Wash buffer D Tube C Add 800 pl Wash buffer E Tube D Add 120 ul Elution Buffer D F Insert the tubestrip tray to the instrument and insert the tip combs into the slots G Close the front lid and start the process by selecting protocol InviMAG_Forensic_mL using arrow keys and press START H Remove the tubestrip tray from the KingFisher mL after the program has completed Important Notes 1 After finishing the extraction protocol the Tube E contains the extracted DNA Store the DNA under adequate conditions We recommend to transfer the extracted DNA into the 1 5 ml Receiver Tubes for further storage and freeze the DNA at 20 C 2 If the DNA contains carryover of magnetic particle
9. soft com 2 Vers 1205 Protocols Isolation of DNA from forensic samples 1 Sample Lysis A Isolation of DNA from Buccal Swabs Transfer 600 ul of Lysis Buffer G and 25 ul of Proteinase K into a 1 5 ml Reaction Tube Transfer the swab into the so prepared tube vortex the tube for 5 s and incubate the sample at 56 C for 20 minutes under continuously shaking e g by using a thermomixer Important Note To get maximum yield of DNA it is essential to leave the swab during the complete lysis time into the reaction tube It is possible to cut the shaft of the swab so that you can close the cap of the reaction tube It is also possible to do the lysis step with opened cap The removing of the swab from the reaction tube ahead of time will result in a dramatically reduced final yield After lysis time carefully squeeze out the swab on the wall of the tube and discard the swab Transfer the lysed sample into the Tube A of the KingFisher tube strip and add 300 ul of Binding Buffer T and 20 ul MAP Solution A see also below Vortex the tube MAP Solution A vigorously before use B Isolation of DNA from blood stains saliva stains etc Cut the material containing the stains into small pieces and transfer it into a 1 5 ml Reaction Tube Add 600 ul of Lysis Buffer G and 25 ul of Proteinase K For semen stains at aditional 30 ul 1 M DTT not provided to the Lysis Buffer and Proteinase K mix Vortex the tube for 5 s Incubate the sample at 56
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