mag maxi kit bulk kit
Contents
1. mag maxi kit pulk kit For in vitro diagnostic use 43301 Z 2500 May 2014 LGC Genomics GmbH Ostendstr 25 TGS Haus 8 12459 Berlin e Germany Tel 49 0 30 5304 2200 Fax 49 0 30 5304 2201 CE li ull Intended use of the mag maxi kits The mag maxi kits were developed to isolate human genomic DNA gDNA from whole blood This can be done manually according to the protocol in this user manual The application of the kit on automated liquid handling devices or automated magnetic bead manipulators is possible as well see recommendation to automate the kit protocol mag maxi kits isolate more than 95 of the gDNA of the sample To isolate DNA from other species e g pathogens bacteria the chemistry must be reevaluated The isolated gDNA can be used as starting material for PCR Polymerase Chain Reaction based analysis For information on protocols for other starting materials please contact our application specialists via email extraction lgcgenomics com or Tel 49 0 30 5304 2250 Index of contents Intended use of the mag maxi kits Symbols Principle of extraction Kit uses Yield and quality Intended user of the mag maxi kits Kit content Storage Safety information Reagent preparation Manual protocol Tips for manual protocol Usage of the mag maxi kits on automated lab equipment Tips for automated protocol adaption Usage of magnetic particle manipulators sep boxes Troubleshooti2. Safety information e Wear appropriate skin and eye protection during the usage of the mag maxi kits e Lysis buffer BLM mag particle suspension BLM and Wash buffer BLM 1 contain high concentrations of salts and detergents Note In case of accidental contact thoroughly rinse or flush the affected areas with water e Prepared Wash buffer BLM 2 contains 70 acetone or ethanol Keep away from naked flames Hazard Hazard Kit component Precaution phrases Guanidine H302 H315 P280 P305 P351 P338 P3 Lysis buffer BLM hydrochloride H319 62 P301 P312 P332 P313 arning ease Proteinase K D H315 H319 P261 P305 P351 P338 lyophilized H334 H335 P342 P311 anger nese m sqa PZOO P303 P361 P383 suspension BLM mocyanate Danger P338 P310 P405 m H302 H312 P260 P303 P361 P353 Wash buffer BLM 1 oe D H332 H314 P305 P351 P338 P310 y Danger H412 P405 Wash buffer BLM 2 concentrate Elution buffer BLM SDS Safety data sheet are available at our Genomics Resource Center on our webpage www lgcgroup com genomics Do not use bleach for decontamination of liquid waste from extraction with mag maxi kit Guandine thiocyanat present in Lysis buffer BLM mag particles and Wash buffer BLM 1 could form harmful compounds with bleach The waste which will be generated during extraction might still be infectious Therefore it is recommended to handle it like infectious waste and dispose it with appropriate safety precautions Reagent pr
3. aporation Alternatively you can use ethanol 96 100 instead of acetone for the preparation of Wash buffer BLM 2 Manual protocol 1 14 15 16 Ensure blood samples are well mixed prior to starting the protocol This is absolutely necessary to ensure effective re suspension of DNA containing parts of the blood sample Add 200 uL of Lysis buffer BLM and 20 uL of Protease to 200 uL of blood sample Mix thoroughly set pipette volume to 350 uL and pipette up and down 5 times Incubate at 55 C for 10 minutes then allow to cool to room temperature Add 200 uL of ethanol to each sample Ensure the mag particle suspension BLM is fully re suspended Add 20 uL to each sample Mix thoroughly set pipette volume to 550 uL and pipette up and down 5 times Incubate for 2 minutes at room temperature to allow sufficient time for binding to occur For agitating the sample during this time use a shaker or vortex periodically Bring magnet into contact with the sample tubes Wait for 1 minute at room temperature to allow the mag particles to form a pellet Remove the supernatant and discard Ensure as much of the supernatant is removed as possible without dislodging the particle pellet Move the magnet away from the sample tubes Add 720 uL of Wash buffer BLM 1 and re suspend the pellet Mix thoroughly set pipette volume to 650 uL and pipette up and down 5 times or until pellet is fully re suspended Incubate at room temperature for 10 mi
4. ation Follow the manual protocol as specified overleaf in respect to working steps incubation times and volumes Tips on automated mixing are given below Mixing with automated liquid handling system e Set mixing volume to be between 50 to 80 of the volume to be mixed instrument dependent e For each mixing step aspirate and dispense between 5 and 10 times depending on the efficiency of the liquid handler e Keep mix aspirate and dispense speeds low with Lysis buffer BLM to avoid frothing e Increase aspirate and dispense speeds when re suspending pellets in wash buffers to ensure complete re suspension Usage of magnetic particle manipulators sep boxes e sep boxes are computer driven magnetic particle collectors manufactured by LGC Genomics with active cooling and heating functionality e Note sep 72 x 1 4 has a maximum working volume of 1 mL e The magnets can be placed in three positions in relation to the sample left right and underneath away from the sample e For effective re suspension of particle pellets it is recommended to move the magnets from the left to right positions using the cycle mode See sep box operating manual for more details e For efficient elution of the nucleic acids from the particles it is recommended to use the cycle mode during the elution incubation period Troubleshooting Problem Possible cause Corrective action PCR inhibition Low yield Coloured eluates Par
5. eparation Presence of precipitates Salt precipitates can form in Lysis buffer BLM mag particle suspension BLM and Wash buffer BLM 1 at low temperatures Check for the presence of precipitates prior to use and if required re dissolve them by incubating the reagents at 37 C for about 10 minutes Protease Prepare the Protease by adding 56 mL of pure water to the vial of Protease When not in use store the Protease at 20 C It is recommended to divide protease solution into suitable aliquots and store at 20 C Lysis mix To reduce the number of pipetting steps a lysis mix can be prepared at the start of the process Thaw the Protease thoroughly Add 20 uL of Protease to 200 uL of Lysis buffer BLM for the number of samples to be processed The table below gives some example calculations including a 10 wastage factor Mix thoroughly Use the lysis mix immediately Number of samples Vol of Lysis buffer BLM Vol of Protease 1 220 uL 22 uL 20 4 4 mL 440 uL 72 15 mL 1 5 mL 96 21 mL 2 1 mL mag particle suspension BLM The mag particles are suspended in a specially formulated buffer which avoids rapid sedimentation or clogging of particles during handling Mix the suspension thoroughly before use to fully re suspend the particles Wash buffer BLM 2 To prepare 100 mL of Wash buffer BLM 2 add 70 mL of acetone to 30 mL of Wash buffer BLM 2 concentrate Mix well Ensure the lid is closed tightly when the bottle is not in use to avoid ev
6. he kit starting gDNA extraction from healthy patients Please note gDNA yield depends on many factors The health conditions of the patient history of the sample storage condition pre extraction treatment influence final yield of extraction and are out of the control of the manufacturer of the kit Intended user of the mag maxi kits gDNA extraction must be carried out in appropriate laboratory environment which is designed for working with sample material having human origin The user of the kit must have been educated in general treatment of sample material of human origin Always wear appropriate protection gloves a lab coat and suitable eye protection during the work with the mag maxi kits Kit content Cat 43301 Lysis buffer BLM Blue 550 mL Protease Grey 1120 mg mag particle suspension BLM White 58 mL Wash buffer BLM 1 Red 2000 mL Wash buffer BLM 2 concentrate Yellow 1200 mL Elution buffer BLM Black 550 mL Handbook Li 1 Additional required reagents e Ultra pure sterile water e Ethanol 96 100 e Acetone Additional buffers can be purchased separately catalogue numbers available on request Storage Kit components should be used within six months of delivery and stored under the recommended conditions Please refer to the kit box label for the expiry date Room temperature 20 25 C Lysis buffer BLM mag particle suspension BLM Wash buffer BLM 1 Protease Wash buffer BLM 2 Elution buffer BLM
7. he walls of the tube and then aspirate a second time to remove these remnants of liquid When removing supernatants it is important to remove as much of the liquid as possible without dislodging the particle pellet With magnets used for manual protocols the particle pellet forms on the back wall of the sample tube When placing the pipette tip inside the tube be sure to aim the end of the tip to the front wall of the sample tube to avoid disrupting the particle pellet Usage of the mag maxi kits on automated lab equipment liquid handling systems and or magnetic particle manipulators The result of gDNA extraction strongly depends on the setup and technical features of the automated lab equipment in use In addition the volumes given in the description of manual protocol have to be adapted to the technical specifications of the robot system in use and might be different transport volume air gap excess volume Even in case of this equipment is labelled as CE IVD device the whole application mag maxi kits and programmed method for DNA extraction must be evaluated regarding the following parameters e Yield and quality of the DNA e Cross contamination of samples with reagents other samples e Transport of samples sample management tracking LGC Genomics offers support in method validation evaluation and how to adapt the protocol of this manual to automated systems extraction lgcgenomics com Tips for automated protocol adapt
8. ng Symbols In vitro diagnostic medical VD device Manufacturer Catalogue number Batch code Consult instructions for use a 2 FE oxen O O O O N OT fF A OO O O WwW DPS eo Contains sufficient for lt n gt tests Use by Do not reuse Temperature limitation Irritant Principle of extraction mag maxi kits use DNA binding magnetic microparticles for the preparation of genomic Deoxyribonucleic Acids gDNA from human whole blood Superparamagnetic microparticles coated with mag surface chemistry are used to capture gDNA from a lysed blood sample The gDNA particle complex is subsequently washed to remove impurities The gDNA is then eluted from the particles and ready for use in PCR based downstream processes Kit uses mag maxi kits are used to extract DNA from whole blood The method was developed and optimised using 200 uL of fresh or frozen whole human blood The following anticoagulants have been tested and found to be compatible with mag maxi extraction kits e EDTA e Citrate In case you want to extract gDNA from other sample material or blood samples preserved with other anticoagulants please contact the manufacturer of the kit for consultation and information Yield and quality Average yield of gDNA using the mag maxi kits is between 4 6 ug UV measurement normally results in a 260 280 ratio which mirrors contamination with proteins gt 1 75 Data are given based on manual application of t
9. nutes agitating the sample during the time period Use a shaker or vortex periodically Bring magnet into contact with the sample tubes Wait for 1 minute at room temperature to allow the mag particles to form a pellet Remove the supernatant and discard Ensure as much of the supernatant is removed as possible without dislodging the particle pellet Repeat steps 9 to 13 with 720 uL of Wash buffer BLM 2 Repeat steps 9 to 13 a second time with 720 uL of Wash buffer BLM 2 Dry the pellet at 55 C for 10 minutes Sample tubes must be left open to allow evaporation to occur Add 200 uL of Elution buffer BLM and re suspend the pellet Mix thoroughly set pipette volume to 150 uL and pipette up and down 5 times or until pellet is fully re suspended Incubate at 55 C for 10 minutes agitating the sample during the time period Use a heated shaker or vortex periodically Bring magnet into contact with the sample tubes Wait for 3 minutes at room temperature to allow the mag particles to form a pellet Remove the eluate and place into a new sample tube To avoid particle transfer it is recommended to transfer only 180 uL of the eluate Tips for manual protocol For manual testing of the protocol or if no magnet is available it is recommended to spin tubes for 10 seconds to enable the magnetic particles to form a pellet y To remove as much liquid as possible it is recommended to aspirate once let any liquid run down t
10. on time to allow time for a tighter pellet to form Position tip further away from pellet whilst removing supernatants Check activity of protease Acetone has a maximum UV absorbance at 268 nm and a Azeo Azgo ratio of 1 53 If this phenomenon occurs prolong the drying time to ensure all the acetone evaporates www lIgcgroup com genomics Email genomics lgcgroup com Ostendstr 25 TGS Haus 8 12459 Berlin Germany Tel 49 0 30 5304 2250 Fax 49 0 30 5304 2201 Units 1 amp 2 Trident Industrial Estate Pindar Road Hoddesdon Herts EN11 OWZ UK Tel 44 0 1992 470 757 Fax 44 0 1438 900 670 100 Cummings Center Suite 420H Beverly e MA 01915 USA Tel 1 978 232 9430 Fax 1 978 232 9435 GO VLO0c L0 v LOSEr All trademarks and registered trademarks mentioned herein are the property of their respective owners All other trademarks and registered trademarks are the property of LGC and its subsidiaries Specifications terms and pricing are subject to change Not all products are available in all countries Please consult your local sales representative for details No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying recording or any retrieval system without the written permission of the copyright holder LGC Limited 2014 All rights reserved 3927 LB 0414
11. ticles present in eluates Low ratio between A2650 and A280 Incomplete buffer removal Poor protease activity Inefficient binding Wash buffer BLM 2 acetone composition lt 70 Incomplete buffer removal Low protease activity Heavily stained sample material Aspirating too fast Loose pellet Disrupting pellet during aspiration Inefficient lysis Acetone carryover in eluate Ensure all the buffer is removed before adding the next buffer Check and if necessary adjust the liquid handling parameters for automated systems Prepare the protease as detailed in the Reagent preparation section aliquot into several tubes and store 20 C Remove and thaw aliquots as required Do not use protease which has been kept at room temperature for an extended period of time e g overnight Ensure that the lysate ethanol and mag particles are mixed thoroughly Ensure that the Wash buffer BLM 2 bottle is closed tightly when not in use to prevent evaporation Ensure all the buffer is removed before adding the next buffer Check and if necessary adjust the liquid handling parameters for automated systems Make sure that protease solution is stored frozen at 20 C protease tends to self digestion which deactivates the enzyme during longer storage at room temperature Check incubation time and temperature Contact our technical specialists for advice Reduce the speed at which supernatants are removed Increase separati
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