LysoBead Direct-to-PCR Extraction Kit

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1 Kit Primerdesign TM Ltd genesig Easy LysoBead Direct-to-PCR Extraction Kit 50 extractions Universal kit for isolation of DNA from meat, water, clinical, veterinary and other samples types genesig Easy LysoBead DNA Extraction Kit 1

2 1 Quick guide 1.1 Quick guide Principle of LysoBead The genesig Easy LysoBead direct-to-pcr Extraction Kit allows for the quick preparation of DNA samples suitable for down-stream molecular applications such as qpcr. Reliable DNA extraction can be obtained from the smallest amounts of source material (up to 20mg or 100µl). The protocol incorporates mechanical disruption technology into a PCR-ready lysis formulation, allowing for a single tube preparation of DNA samples for molecular testing in under 10 minutes. This is a significant reduction in the time taken to extract DNA from microbes with highly developed cell walls, such as gram-positive bacteria, fungal and mycobacteria samples, where the lysis stage was typically augmented with either a prolonged overnight incubation, or with mechanical disruption steps. Suitable sample types: Clinical including swabs and urine Veterinary Environmental including plant, soil and water Suitable to isolate DNA from pathogens: Bacteria Viruses Yeast and Mycobacterium SAMPLE LysoBead 1. Vortex 2. Heat 3. Add directly to PCR genesig Easy LysoBead DNA Extraction Kit 2

3 1.2 Quick guide Sample Prep Depending on your sample type you should add different quantities of sample, you may also need to perform a simple sample prep step to prepare for DNA extraction. Swab samples Add swab directly to LysoBead and leave for 10-30secs Liquids (excluding blood/serum/plasma) Add 100µl sample directly to the LysoBead tube Solids (excluding meat/soil) Add a 10-20mg (a match stick head in size) sample directly to LysoBead tube Meat Add < 5mg (pipette tip head) of tissue directly to LysoBead tube Soil / Plant material Combine Approx mg of faeces/soil (a match head in size) or 200µl if using liquid faeces 500µl Sample prep solution* Mix well with pipette and leave to settle out Add 100µl clear top layer of liquid to LysoBead tube If you are concerned that you sample doesn t fall within one of the above categories, or is a high-fat content sample then please contact us at enquiries@primerdesign.co.uk for advice on sample preparation. *Sample preparation solution must be purchased separately genesig Easy LysoBead DNA Extraction Kit 3

4 1.3 Quick guide LysoBead DNA extraction See main body of handbook for detailed instructions of use, tips and troubleshooting genesig Easy LysoBead extraction Step Instructions 1 Add sample to LysoBead tube 2 Add 10µl genesig Easy Internal extraction control DNA 3 Vortex Maximum speed for 5 min 4 Heat 80 C for 2.5 min 5 Centrifuge < 5,000 g for 1 min 6 Transfer 200μl of solution to a fresh tube DNA is in the liquid! If using a standard or advanced genesig kit, use 5µl of the internal extraction control template. genesig Easy LysoBead DNA Extraction Kit 4

5 2 Components 2.1 Kit contents 50 x LysoBead tubes (50 extractions) If using the sample LysoBead kit this will contain 10 x LysoBead tubes (10 extractions) 2.2 Additional items If working with soil or plant materials sample preparation solution must be purchased separately. This can be purchased from or by contacting us at enquiries@primerdesign.co.uk genesig Easy LysoBead DNA Extraction Kit 5

6 3 Product information 3.1 Intended use and principles of the procedure The LysoBead direct-to-pcr extraction kit is intended for the rapid and easy manual extraction of DNA from a range of sample types. Each LysoBead tube contains the glass beads and buffer required for DNA isolation from 1 sample. The whole procedure is divided into three steps: (i) cell lysis with the aid of glass beads, (ii) incubation at elevated temperature, and (iii) removal of cell debris by centrifugation. The extracted genomic DNA may directly be used for downstream applications and can be stored at -20 C. 3.2 Reagents and equipment to be supplied by the user Heat block capable of reaching 80 C Vortex Genie or a stand-alone vortex Table top microfuge/centrifuge Disposable gloves Timer Pipettes Microcentrifuge tubes 4 Storage conditions Store all kit components at 2-8 C. Do not use the reagents beyond their expiry date. Dispose of unused reagents and waste in accordance with federal, state and local regulations. Always wear suitable protective clothing and gloves. Resulting LysoBead samples should be stored at -20 C for future use. genesig Easy LysoBead DNA Extraction Kit 6

7 5 Protocols 5.1 Preparation of sample materials a) Swab samples Samples in swab form should be thoroughly eluted in the LysoBead tube for seconds and then discarded. Proceed with LysoBead treatment. b) Liquids (excluding blood/serum/plasma) Add up to 100µl of sample directly to the LysoBead tube and proceed with LysoBead treatment. c) Solids (excluding meat/soil) Add up to 20mg of sample directly to the LysoBead tube and proceed with LysoBead treatment. d) Meat samples No more than 5mg of sample material should be added directly to the LysoBead tube. Quantities greater than this will introduce significant amounts of PCR inhibitors into your PCR reaction. Proceed with the LysoBead treatment. e) Fish samples Add up to 20mg of sample directly to the LysoBead tube and proceed with LysoBead treatment. f) Soil/plant material Soil and plant material should be subjected to a sample preparation step. Approx mg of soil/plant material should be added to 500µl of Sample prep solution*. Mix well with a pipette. Leave to settle out. Add 100µl clear top layer of liquid to LysoBead tube. Proceed with LysoBead treatment. * Sample preparation solution must be purchased separately genesig Easy LysoBead DNA Extraction Kit 7

8 5.2 LysoBead treatment of DNA from meat, water, clinical, veterinary and other samples types 1 Addition of Sample Add sample to LysoBead tube according to 5.1 Preparation of sample materials Top tip For tissue samples ensure that a minimum amount of blood is added to the LysoBead tube 2 Vortexing Subject the LysoBead tube (containing sample) to a 5 minute vortex at maximum speed 3 Heating Transfer your sample to a pre-heated heat block for 2.5 minutes at 80 C 4 Centrifugation Spin down the resulting sample for 1 minute at <5,000 x g and carefully remove the tube from the centrifuge 5 A Fresh Tube Transfer a minimum of 200µl of the solution to a fresh microcentrifuge tube (being careful to avoid the glass beads and any possible sample debris). The sample can be added directly to PCR reaction at 1-10µl per reaction or can be stored at -20 C for future use. Detailed protocol This protocol is for manual use with the LysoBead extraction kit. genesig Easy LysoBead DNA Extraction Kit 8

9 6 Safety instructions Please follow the safety instructions on the MSDS. genesig Easy LysoBead DNA Extraction Kit 9

10 7 Appendix 7.1 Troubleshooting Problem Possible cause and suggestions Absence of amplification in PCR DNA solution contains inhibitors. Ensure appropriate starting material and swab system. If problems persist, the issue could be caused by improper sampling, storage, transport, or preparation of specimen. Request new specimen and repeat DNA extraction. Too much sample added to LysoBead tube Compromised amplification traces Try adding less sample to the LysoBead tube or carrying out a sample preparations step prior to the extraction if the sample is in solid form. If using a sample preparation step, add up to 100μl of the resulting solution to the LysoBead tube for extraction. Pieces of sample transferred to PCR Poor purity Make sure to centrifuge the sample after the extraction procedure has been carried out, taking care to avoid large sample debris when transferring to a fresh tube. Low sensitivity DNA solution contains protein contaminations. Extend centrifugation step and transfer supernatant (DNA solution) to a new tube. Centrifugation or time for beads to settle too short Carry-over of beads Increase centrifugation time to allow the beads to settle completely before transferring the resulting sample to a fresh tube for downstream applications. genesig Easy LysoBead DNA Extraction Kit 10

11 7.2 Ordering information Visit for more detailed product information. 7.3 Product use restriction / warranty genesig Easy LysoBead extraction kit components are intended, developed, designed, and sold FOR RESEARCH PURPOSES ONLY, except, however, any other function of the product being expressly described in original PRIMERDESIGN product leaflets. PRIMERDESIGN products are intended for GENERAL LABORATORY USE ONLY. PRIMERDESIGN products are suited for QUALIFIED PERSONNEL ONLY. PRIMERDESIGN products shall in any event only be used wearing adequate PROTECTIVE CLOTHING. For detailed information please refer to the respective Material Safety Data Sheet of the product. PRIMERDESIGN products shall exclusively be used in an ADEQUATE TEST ENVIRONMENT. PRIMERDESIGN does not assume any responsibility for damages due to improper application of our products in other fields of application. Application on the human body is STRICTLY FORBIDDEN. The respective user is liable for any and all damages resulting from such application. DNA purification products of PRIMERDESIGN 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 (including, but not limited to diagnostic, prognostic, therapeutic, or blood banking). It is rather in the responsibility of the user or - in case of resale of the products - in the responsibility of the reseller to inspect and assure the use of the DNA/RNA purification products of PRIMERDESIGN for a well-defined and specific application. PRIMERDESIGN shall only be responsible for the product specifications and the performance range of PRIMERDESIGN products according to the specifications of in-house quality control, product documentation and marketing material. Documentation stating specifications and other technical information can be found at PRIMERDESIGN warrants to meet the stated specifications. PRIMERDESIGN 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 PRIMERDESIGN. Please contact us if you wish to get an extra copy. There is no warranty for and PRIMERDESIGN is not liable for 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; defects in products or components not manufactured by PRIMERDESIGN, or damages resulting from such non-primerdesign components or products. PRIMERDESIGN 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 PRIMERDESIGN PRODUCTS. In no event, shall PRIMERDESIGN be liable for claims for any other damages, whether direct, indirect, 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 PRIMERDESIGN products to perform in accordance with the stated specifications. This warranty is exclusive and PRIMERDESIGN makes no other warranty expressed or implied. The warranty provided herein and the data, specifications and descriptions of this PRIMERDESIGN product appearing in PRIMERDESIGN published catalogues and product literature are PRIMERDESIGN s sole representations concerning the product and warranty. No other statements or representations, written or oral, by PRIMERDESIGN s employees, agent or representatives, except written statements signed by a duly authorized officer of PRIMERDESIGN 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. Product claims are subject to change. Therefore, please contact our Technical Service Team for the most up-to-date information on PRIMERDESIGN products. You may also contact your local distributor for general scientific information. Applications mentioned in PRIMERDESIGN literature are provided for informational purposes only. PRIMERDESIGN does not warrant that all applications have been tested in PRIMERDESIGN laboratories using PRIMERDESIGN products. PRIMERDESIGN does not warrant the correctness of any of those applications. Last updated: 14/10/2014 Please contact: PRIMERDESIGN Ltd support@primerdesign.co.uk Trademarks: Primerdesign TM a trademark of Primerdesign Ltd genesig is a registered trademark of Primerdesign Ltd All used names and denotations can be brands, trademarks, or registered labels of their respective owner also if they are not special denotation. To mention products and brands is only a kind of information (i.e., it does not offend against trademarks and brands and cannot be seen as a kind of recommendation or assessment). Regarding these products or services we cannot grant any guarantees regarding selection, efficiency, or operation. genesig Easy LysoBead DNA Extraction Kit 11