User s Manual. Bacteria Genomic DNA Kit. ExiPrepTM Bacteria Genomic DNA Kit K Version No.: 3.0 ( )

Transcription

1 ExiPrepTM Bacteria Genomic DNA Kit ExiPrepTM Bacteria Genomic DNA Kit Kit for the samples extraction using of bacteria ExiPrepTM genomic instrument DNA in various K-3245 Version No.: 3.0 ( ) User s Manual BIONEER 49-3 Tel: Fax: Munpyeong-Dong, CORPORATION order@bioneer.co.kr Daedeok-Gu, Daejeon, , Korea

2 Safety the Warnings and Precautions Manual, Please Before, Material all inquire during potentially Safety BIONEER s and Data hazardous after Sheet Customer use (MSDS) of materials this Service for kit this (i.e. as Center product. materials described to obtain that may a this copy have User of come should appropriate in be contact processed regulations with clinical and of the disposed samples) municipality/government including of according tubes, to tips applicable in and which materials and integrity product Please is read being the used. User Manual before using this Kit. Please check this to the use. experiment. Adhere of all tubes, to general tips and clinical other laboratory materials supplied safety procedures with this kit during prior the Warranty control BIONEER All BIONEER and products Liability during the protocols guarantees warranty certified period the are quality under of manufactured one of ISO (1) all year 9001 directly and from and tested date manufactured ISO under of purchase. strict standards. products quality issues immediately (order@bioneer.com). are discovered contact relating BIONEER s to compromise Customer in Service product quality, Center If any Trademarks ExiPrepTM Copyright is the trademark 2011 by Bioneer of Bioneer Corporation. Corporation. All KOREA rights reserved KIT COMPONENTS STARTING INTRODUCTION STORAGE Table SAMPLE&TYPICAL of Contents GENOMIC Additionally DNA required EXTRACTION materials &... YIELD devices Extraction from urine sputum cultured... bacteria PROTOCOL TROUBLESHOOTING Extraction from NUMBER swab... LIST ORDERING INFORMATION EXPLANATION REFERENCES... OF SYMBOLS 17 18

3 Kit components ExiPrepTM Bacteria (K-3245) Genomic DNA Kit Buffer Cartridge Resuspension Disposable Elution Tip Buffer 3 ea User s Tube (8-strip) One Page Manual Protocol 1 pack ea (including Storage within ExiPrepTM binding/washing/elution Kits are designed to contain buffers) all and necessary components buffers Bacteria Genomic DNA Kit protective the Buffer film Cartridges. to prevent All Buffer leakage, Cartridges evaporation are sealed and 2. contamination. temperature without (15-25 ) The Buffer under Cartridges dry conditions can be for stored up to at 2 room ExiPrepTM opening. Kits may also provide enzymes in lyophilized years for Cartridges Lyophilized increased and/or convenience. enzymes 2.0mL may screw be cap contained tubes. within The enzymes the Buffer form lyophilized 25 ) Reconstituted up to form 2 enzymes years can be without must stored be any stored at detectable room at -20 temperature loss in activity. (15 - in are consumables The DNase disposable and and RNase tips avoid and free. nuclease elution Please contamination. tubes take included caution in within storing the the Kit 1 2

4 extraction bacteria ExiPrepTM Introduction undergo and of Bacteria genomic fungi. Gram Genomic DNA positive from DNA gram bacteria Kit negative is designed and and fungi positive for must the Bacteria Genomic DNA Kit The digested enzymatic samples them digestion must step be for resuspended optimal extraction. with the 3. provided DNA Cartridge Samples extraction. Resuspension are mixed with Buffer Lysis before Buffer proceeding contained with within genomic be released 1 into and the undergo solution. incubation After the so incubation the nucleic step, acids Buffer magnetic will be introduced to bind the DNA. silica may (containing are then washed beads cellular to can debris) flush be any through impurities separated magnetization. that from may the exist. The solution Finally, beads The a beads form small and will volume is release ready of the to elution be DNA. used. The buffer eluted washed DNA over is in the a highly magnetic pure [Description of DNA extraction using silica magnetic bead] 4 and application Typical Starting the typical amounts Sample yields of starting are & Typical described material yield below. quantity, Results elution of your volume Sample may type vary. Starting Sample Volume Elution Typical Yield 4. Gram (S.(+) Yeast (-) bacteria Fungi pombe) ~1x108cells 50~200ul Genomic 5-15ug Disposable DNA gloves Extraction Pipettes Sterilized 1.5ml microcentrifuge pipette tips with tubes filters or 15ml conical tubes x Lysozyme Table PBS top buffer, centrifuge or lyticase 1x TE buffer, or swing 10N rotor NaOH ExiprepTM 16 Pro Plus (Cat. No. No. : A-5040, : A-5030, Bioneer centrifuge Co.,KOREA), 5.1 Additionally required materials & devices 3

5 5.2 Extraction from cultured bacteria This from protocol cultured is bacteria. 1. designed a. for extraction of genomic DNA ExiPrep TM Bacteria Genomic Resuspend For Gram (-) bacteria DNA Kit cells) in 200ul the of cell Resuspension pellet (up Buffer. to 1x108 b. cells) Resuspend Add For Gram 20ul 200ul of the (+) of lysozyme bacteria 1x cell TE pellet buffer (50mg/ml) (up to 1x108 incubate 1hr. Centrifuge the the tube tube at rpm for at least and 3min - Resuspension Re-suspend and remove Buffer. the the pellet supernatant. with 200ul for of 2. Place and tray. Buffer the elution Cartridge tube 1 rack, on the tip setup rack, 3. Ensure that all tips and tubes are ExiPrep TM 4. inserted Punch in holes the desired through positions. the Buffer Bacteria Genomic DNA Kit Cartridge punch corresponding Before punching tool sealing to the in film the tubes holes, with the and the make locations tips. 6-hole to suspend shake Buffer Cartridge 1 sure outward the to beads, settle then solutions gently to swing the to 5. Load loading bottom 200 wells. of μl the of wells. sample into the sample Turn on the ExiPrepTM 8. the Store button instrument PREP Press (ExiPrepTM SETUP the Start 16 menu. Pro only) button for to cooling. access the 5 6

6 9. Enter a protocol number from the ExiPrep TM Bacteria Genomic 10. protocol your acid type. sample list (Chapter source and 7) that target best nucleic suits DNA Press Kit 11. the Select next the the step. Enter desired button elution to proceed volume to 12. from Press Recommended the the screen. ok button elution to volume: move to 100ul 13. next Open step. the instrument door and pull the 14. out Place their Plate the all Base racks Plate. (steps correct according 15~19). positions and to Buffer the on CHECK Cartridges the Base LIST in 15. Place labeled Buffer position Cartridge on the Base 2 Plate. into its 16. Place labeled Buffer position Cartridge on the Base 1 Plate. into its 17. Place a. Place labeled For the the ExiPrepTM position Elution on Tubes 16 Tube the Plus in Base Rack Lanes Plate. into A and its B b. of For the ExiPrepTM Elution Elution 16 Tubes Pro Rack. 18. and Place 3rd the rows Disposable of the Elution Filter into Tube Tip the Rack. 2nd into Plate. its labeled position on the Base Place position and Push 2). the (between Waste Tray Buffer into Cartridges its proper into the Base instrument Plate completely and close back the 1 door. 7 8

7 21. Verify screen. the next target the The nucleic first protocol two acid letters name type, represent and on the ExiPrep TM Bacteria Genomic DNA source. two letters represent the sample Kit Press extraction When the the run. Run process button is to successfully start the 24. completed, Open door three and options pull out will display. 25. Take the Elution Tubes from the 26. Plate. Remove the Buffer Cartridges and Base 27. racks door. Quantify from the Base purified Plate genomic and close DNA the all desired). (if 5.3 Extraction from urine This DNA protocol from urine 1. is Transfer designed samples. for the extraction of genomic ExiPrep TM Bacteria Genomic a 15ml an adequate volume of urine into DNA Kit 2. Centrifuge conical 3,000 tube. 3. Add is Please insufficient. 200μl repeat of Resuspension centrifugation rpm for 20 Buffer if min. pelleting If the mix the volume well sample by vortexing of has Resuspension excessive for 15 sedimentation, sec. Buffer may and be increased up to 500μl. 4. Go cultured during Tip: to These the step bacteria. sample 2. steps of centrifugation 5.2 may Extraction be prepared step. from 9 10

8 5.4 Extraction from sputum This DNA protocol from sputum 1. is designed Add samples. for the extraction of genomic ExiPrep TM Bacteria Genomic container 1/10 volume of 10N NaOH into the DNA Kit and the mix sample by holding vortexing tubes for the for 15 sputum 3 min. Incubate sample temperature. 10N If sample has high viscosity, add room sample NaOH tube (up for to up 500μl) to 30 or min. incubate more the Transfer microcentrifuge Centrifuge 1.5ml the of tube. sample at 9,000 into rpm a 1.5ml min supernatant. If your and completely is remove incomplete, for the 5 please repeat centrifugation Re-suspend PBS Centrifuge min buffer. the pellet with 1ml of 1X ExiPrep TM Bacteria Genomic supernatant. and the completely tube at 9,000 remove rpm for the 5 DNA Kit Repeat Resuspend Resuspension step 4.~5. the Buffer. pellet over two with times. 200μl of 8. Go cultured to step bacteria. 2. of 5.2 Extraction from 11 12

9 5.5 Extraction from swab This DNA protocol from swab 1. is Transfer designed samples. for the extraction of genomic ExiPrep TM Bacteria Genomic tube the swab into a 15ml conical DNA or Kit 2. Add and 1ml a of specialized 1x PBS buffer sample or tube. media, If the mix specialized well do not by vortexing. add sample the 1ml tube nomal liquid contains saline 3. Transfer proceed 500μl to the of next sample step. into a 1.5ml and microcentrifuge Centrifuge min Resuspend the tube. Resuspension and remove the Buffer. the pellet supernatant. 13,000 with rpm 200μl for of 2 6. Go cultured to step bacteria. 2. of 5.2 Extraction from 6. Troubleshooting A. Low yield of Genomic DNA 1) Did ExiPrep TM you add sufficient amount of sample? The yield is Bacteria Genomic DNA Kit 2) dependent Overloading sample on the may sample sometimes type decrease and the amount. 3) Did decreases Is there you salt lyse yield precipitate the and samples purity. in any completely? of the buffers? Incomplete Warm yield. lysis 4) bottles Did Incomplete you at 60 shake resuspension to re-dissolve. Buffer of cartridge the magnetic 1 before beads use? may the decrease the yield and purity. DNA particles will The magnetic after cannot genomic particles bind DNA nucleic may extraction. acid co-elute in Elution Co-eluted with the Buffer magnetic extracted simple Co-eluted not centrifugation. decrease magnetic the yield particles and purity. can easily be separated and by it B. Co-eluted magnetic particles 13 14

10 C. Empty elution tubes 1) Too Please manual. ExiPrep by much starting TM Bacteria magnetic add Excessive the volume sample of volume sample may suggested cause clogging this Genomic beads DNA Kit 2) Insufficient When extraction, processing elimination and samples of interfere the supernatant to with prepare normal extraction. sample Insufficient supernatant you must ensure (ex. Lipid complete layer, removal them medium). for by magnetic removal beads and of interfere impurities with may normal cause extraction. clogging of storage temperatures buffers Please and conditions store force will the the increase (Chapter film Buffer to the bulge. Cartridges 2. vapor Storage). pressure in recommended High of storage certain D. Expansion of sealing film 7. Protocol 1 No. 01 number Genomic Target list DNA Sample Whole blood source ExiPrep TM Bacteria DNA Kit Animal FFPE Plant tissue seed tissue Cultured (+) Rice Gram Yeast (-) bacteria cell Buffy Sputum Fungi Saliva BAL coat Urine Swab Genomic DNA Respiratory Stool CSF EPS sample 15 16

11 Ordering information Product Size Cat. No. Blood K Tissue Bacteria Genomic DNA DNA Kit Kit K-3225 Plant Genomic DNA Kit K-3245 DNA K-3255 ExiPrepTM Viral Beef Rice Genomic RNA Kit DNA Kit 96 prep. K-3200-CR K-3200-CB K-3515 K-3525 of use B., Obata, a Kawamura, novel References K., method Segawa, Y., Yohda, for O., operating Yakabe, M., Matsunaga, magnetic M., Ishida, particles T. Y., and Kuroita, Tajima, magtration T., H. Ikeda, (2001). technology, K., Development Kawakami, and its Sample Honore-Bouakline, J. L. for (2003). automating Rapid nucleic S., Diagnosis Vincensini, acid of purification. Extrapulmonary J. P., Giacuzzo, J. Biosci. Tuberculosis V., Bioeng., Lagrange, 91(5), by P. PCR: H., Herrmann, Impact of 9. Preparation and DNA Extraction. J. Clin. Microbiol. 41: Microbiology (1999) selective Boom, Improved R., binding Sol, 37, C., silica-guanidiniumthiocyanate of Beld, bovine M., Weel, alphacasein J., Goudsmit, to silica DNA J. and isolation particles. Wertheim-Van procedure Journal of based Dillen, Clinical on P ExiPrepTM 16Plus 16Pro 1 ea A-5030 A-5040 Explanation Catalog of symbols Number 10. Contains Batch code Expiration Sufficient Date for test Storage Manufacturer Conditions (Temp.) Caution, Consult accompanying documents

12 Headquarters Address Bioneer ExiPrep TM 49-3, World Wide Bacteria Munpyeong-dong, Genomic DNA Daedeock-gu, Kit Tel Fax Web (Korea only: ) daejeon , Korea Seoul Address Tel Fax Office Web 2F Samhwawa Sansu (korea Building, only: ) 6, Yangjae-Dong, Seocho-Gu, Seoul , Korea Address Bioneer,Inc. Tel Fax Web (Toll Atlantic Avenue Alameda, Free) CA USA Bioneer Address Tel Fax Trade 403Room, (Shanghai) Building 88, Co, no.887, Ltd PuDong New District, Shanghai Zuchongzhi , China Road, Zhangjiang High Tech Park, Web 19