DNA EXTRACTION SERVICES

Transcription

1 DECEMBER 06, 2016 DNA EXTRACTION SERVICES McGill University and Génome Québec Innovation Center DNA Extraction User Guide Version 1.0 Copyright 2016 Centre d innovation Génome Québec et Université McGill Tous droits réservés.

2 Table of Contents TABLE OF CONTENTS... 2 SERVICE REQUEST... 3 SERVICE REQUEST... 3 GENERAL GUIDELINES... 3 SUBMISSION AND SENDING REQUIREMENTS... 4 SAMPLE SUBMISSION FILE... 4 THE FILE MUST BE SAVED BY RENAMING IT AS FOLLOWS: PROJECTNAME_PI(INITIALS OF THE NAME OF PRINCIPAL INVESTIGATOR)-BOX00X (E.G.CHILDBC_YR-BOX001) WHEN THE DOCUMENT IS COMPLETED AS INSTRUCTED, PLEASE SUBMIT IT DIRECTLY INTO YOUR PROJECT IN NANUQ, OR IT TO THE PERSON IN CHARGE OF YOUR PROJECT AT CLIENT MANAGEMENT OFFICE AND /OR TO THE DNA EXTRACTION TECHNICIAN BLOOD AND BUFFY COAT SAMPLES... 6 Media types containers and anticoagulant... 6 Amount of starting material... 6 SALIVA AND BUCCAL SWAB SAMPLES... 6 Type of collection containers accepted... 6 Amount of starting material... 7 CELLS SAMPLES... 7 Type of containers accepted... 7 Amount of starting material... 7 OTHER TYPES OF SAMPLES... 7 MINIMUM PROCESSING SCALE... 8 REQUIREMENTS OF SAMPLE ORGANIZATION... 8 Individual tubes... 8 Plates... 8 SAMPLE PREPARATION FOR SENDING... 9 PREPARATION AND ANALYSIS OF DNA SAMPLES AVAILABLE CONTAINERS FORMATS FOR DNA SAMPLES DNA CONCENTRATION ANALYSIS DNA QUALITY ANALYSIS SAMPLE IDENTIFICATION AND RESULTS FILES FOR MORE INFORMATION CLIENT MANAGEMENT OFFICE

3 Service request Service request We offer a DNA extraction service for several different types of samples like blood, saliva, buccal swab, buffy coat and cell samples. We are continually working to develop various extraction services for many types of samples that are not presented in this document. For any service or to receive information regarding DNA extraction service, please communicate directly with the Client Management Office General guidelines All samples submitted for DNA extraction must be recognized as free from human pathogens. In case samples may contain pathogens, the client must notify the platform manager and mention which pathogens could be present in the sample. Samples that may contain or consist of viruses, bacteria or mold/fungus from Biosafety Level 2 and above are not accepted by our DNA extraction service. Samples of molds and fungi that are known to produce spores are also not accepted because of the risk of contaminating the equipment and the facilities. Usually, we do not accept biomedical samples such as urine, feces and semen. At all times, we reserve the right to change our policies to accept or deny some types of samples. To avoid any delay in processing your request, carefully follow the instructions relating to sample preparation and submission described in this document. Note that the samples are processed on a "first come, first served" basis and that the turnaround time may vary depending on the project size. It is recommended to contact the Client Management Office about processing time when submitting your samples. 3

4 Submission and sending requirements Sample submission file To submit samples for extraction you must complete the document (Excel file) named Model for individual samples. This document is available on Nanuq in the LIMS section, Reception/Submission of samples, Excel template, Template file to download, Chip Validation - Model for individual samples. It is important to save the file in Excel (.xls) format. The Excel file accepts the following characters: a-z, A-Z, 0-9, dot, dash (-), underscore (_) and (@). Please do not use any other character because the file will be rejected by our computer database. Moreover, it is important to limit the sample name (Sample name), the Individual ID and pedigree to a maximum of 25 characters. Required fields are marked with an asterisk (*) or a plus sign (+). Every individual must have an unique individual identification name (Individual_ID). Please complete the following boxes as instructed below: Type: Specify the type of sample according to the choices available in the dropdown menu: Blood, Buffy coat, Cells, Spit (Saliva), Pellet (bacteria/cell), Serum, Swab, Animal or Plant Tissue. One sample type per file is accepted. If you submit several types of sample, you must submit an equal number of sample submission files. Cohort: To be indicated according to your needs, otherwise leave empty. Taxon: Enter the taxon of the species of the sample to extract. Individual name prefix: For internal use only, do not enter anything in this box. Pedigree prefix: For internal use only, do not enter anything in this box. Volume units: Indicate the unit of volume used for liquid samples such as blood, saliva, buffy coat, etc... Concentration units: Leave blank, irrelevant for the samples to be extracted. 4

5 Individual container kind: Select tube in all cases. Container barcode: Please enter the barcode of each individual tube if there is one; otherwise leave it blank. Sample name: Enter the name of the sample using the simplest possible name and only using allowed characters. If several of your samples have the same name, it is appropriate here to add a text item at the end of the name to differentiate, for example _2, or b, etc... Location barcode: For internal use only, do not enter anything in this box. Location coord: For internal use only, do not enter anything in this box. Individual name: Enter the name of the sample using the simplest possible name and only using allowed characters. If several of your samples are from the same individual, it is appropriate here to add the same Individual name for all of the same individual. Gender: Indicate the type by selecting from the dropdown menu. Pedigree: Indicate pedigree of your sample (family, group, mother or father) if the relevant information is known and otherwise just copy the sample name. Mother ID: Identity of the mother (Mother Individual name) if known and relevant, otherwise leave empty. Father ID: Father's Identity (Father Individual name) if known and relevant, otherwise leave empty. Vol.: Indicate the volume of liquid samples according to the unit selected previously. Only numbers are allowed in this column, do not indicate unit after the number. Ideally, you must enter the real volume in the tube and not a theoretical approximative estimate. Indicating an incorrect volume may adversely affect the purity of the DNA obtained. Conc.: Leave blank, irrelevant for the samples to be extracted. Contact ID: Enter the principal investigator Nanuq identifier, or contact Nanuq identifier. Alias: If possible please number your samples from number 1 in their order of presentation in the box/container. This number will be used to identify the tubes during the extraction process of your samples. Site of origin: Indicate the origin of the sample, if relevant. Tissue source: Indicate the source of the tissue, if applicable. Sample blank: Leave blank, irrelevant for the samples to be extracted. Default control: Leave blank, irrelevant for the samples to be extracted. Comment: Indicate any useful comment about the sample. The extraction team often takes notes about the sample at this location when leaved empty. Experimental group: Indicate the experimental group if relevant to the sample. If unused, the extraction team notes the extraction batch, if applicable. 5

6 The file must be saved by renaming it as follows: ProjectName_PI(Initials of the name of Principal Investigator)- box00x (e.g.childbc_yr-box001). When the document is completed as instructed, please submit it directly into your project in Nanuq, or it to the person in charge of your project at the Client Management Office and/or to the DNA extraction personnel. Blood and buffy coat samples We accept all blood and buffy coat samples from animals and healthy donors. The client must notify Génome Québec if samples are coming from sick patients, or may be infected with pathogenic viruses such as HIV virus or Hepatitis C. Media types containers and anticoagulant Samples of blood and buffy coat must contain an anticoagulant such as heparin or EDTA. Samples containing coagulated blood will give a DNA sample contaminated in greater proportion by blood proteins. Clients should avoid sending blood clots or samples containing dried blood. Blood samples can be sent directly in their medical collection tube (Vacutainer type) or be transferred to another type of container with a screw cap that closes tightly. Buffy coat samples must be transferred to an appropriate microtube or cryotube with a screw cap that closes tightly. Amount of starting material The volume of blood accepted varies between 0.5 and 10 ml. The minimum volume suggested is 1mL. It is important to understand that we must use the same initial blood volume for all samples of the same batch. The client must therefore group the samples according to the actual volume of blood in the container. We use the maximum available volume for all samples. Unless otherwise indicated, the residual blood (excess volume relative to other samples) will be discarded following the extraction. As the residual volumes are minimal and all different, it is not possible to reuse them for further extraction. The volume of buffy coat accepted varies between 0.1 and 2 ml. The minimum volume suggested is 0.5 ml. We use all the sample volume. Saliva and buccal swab samples We accept all saliva samples from healthy donors. The client must notify us if samples from patients can be infected by pathogenic viruses such as HIV virus or Hepatitis C. Type of collection containers accepted We accept saliva samples and buccal swabs collected with DNA Genotek products. Clients should use the Oragene or ORAcollect collection kits from DNA Genotek because they must be compatible with the extraction protocol associated to the product Prep-It kit L2P from the same company. GeneFix Saliva DNA Collection Kit from Isohelix should also work. For buccal swabs we recommend using the OCR-100 kit. We cannot guarantee the outcome of DNA extraction if saliva samples are collected with products other than DNA Genotek collection kits or differently than specified by the manufacturer DNA Genotek. DNA Buccal Swabs Kit from Isohelix should also work. 6

7 Amount of starting material According to the collection kit used, the harvested volume of saliva ranges from 0.5 to 3 ml. The minimum volume suggested for obtaining a sufficient amount of DNA is 1mL, lower volumes tend to yield smaller amounts of DNA. Ideally, all collection instructions specified by the manufacturer must be followed to the letter. A supervised collection of good quality typically gives samples with a similar volume of clear and colorless saliva that contains little or no particles. It is important to understand that we must use the same initial volume of saliva for all samples of the same batch. The client must therefore group the samples in batches, depending on the actual volume of saliva in the container. We use the maximum available volume for all samples. Unless otherwise indicated, the residual saliva (excess volume relative to other samples) is discarded following the extraction. As the residual volumes are minimal and all different, it is not possible to reuse them for further extraction. The maximum volume of saliva that can be extracted is 3 ml. Cells samples We accept any type of cell culture that was not used in viral replication and any primary culture from noninfected tissue. Type of containers accepted All cell samples must be sent pelleted in a conical bottom microcentrifuge tube of 1.5mL. Only the pellet must be present at the bottom of the tube, the supernatant must be removed. In some cases, the cell pellets can be sent in a regular half-skirt 96 wells PCR plate. Amount of starting material The amount of cells accepted varies between 1 to 2 million cells. We suggest a minimum of 1 million cells and not to exceed 2 million for optimal results. The sample is completely extracted. Other types of samples We are willing to receive other types of samples according to the availability of a validated extraction method. We are continuously developing new extraction protocols to meet the needs of our clients. We hope to offer a retrieval service soon, covering all samples from animals and plants. Please contact us to know the new types of samples accepted. Important! Do not send samples in microtubes smaller than 1.5 ml tubes or into strip-tubes! 7

8 Minimum processing scale Please note that a minimum of 12 samples is required for the extraction of individual samples in tubes (blood, saliva, buffy coat, buccal smears), otherwise, a minimum of 12 samples will be charged for the lot. For DNA extraction projects of plated samples (digestion only) they will be charged in batches of 96 samples (regardless of the number of samples in the plate). Requirements of sample organization Please pay attention to the following requirements regarding the organization of the samples in boxes, containers or plates for DNA extraction projects. Individual tubes All samples in individual tubes must be organized in logical order in a box or other container that allows to present the samples in the same order as presented in the sample submission Excel file. The samples are presented in the container in the logical order of reading by starting at the top left, towards bottom right. Individual tubes must not be sent packaged individually and unclassified. We reserve the right to add costs for any batch of samples sent individually wrapped or unclassified for the extra work associated with the unpacking and sorting of your samples. Plates The samples are shown in the plate in the logical order of reading. They are organized in rows, starting at the top left in wells A1 to A12, B1 to B12, etc... until the bottom right H12. Do not completely fill the wells as this increases the risk of well to well contamination. Centrifuge your plates and remove the supernatant to send only cell pellets. Use only half-skirt PCR plates. The plates should be properly sealed. Using an adhesive film of good quality such as PCR Adhesive Films from Thermo Fisher Scientific company (Cat # AB-0558). 8

9 Sample preparation for shipping To ensure the preservation of the integrity of the various samples during transport and storage, please follow these instructions. Samples of blood, buffy coat, or cells must be sent on dry ice, classified in their respective containers and properly identified. Samples of saliva and buccal swabs are usually sent at room temperature, classified in their respective containers and properly identified. Each box or container must be packed in a pressure zippered plastic bag (Ziploc type). The name of the Project, the name of the Principal Investigator, the Recipient name and the Platform (biochip-dna extraction) must be clearly indicated on the outside of the box/bag. If samples are sent by regular mail or other delivery service, please use the following address: Attn: Platform-Microarray DNA extraction Daniel Vincent and Rosalie Fréchette McGill University and Génome Québec Innovation Centre 740, Dr.-Penfield, room 7500 Montréal (Québec) Canada H3A 0G1 An should be sent to the recipient when samples are shipped. 9

10 Preparation and analysis of DNA samples Available containers formats for DNA samples Following extraction, your samples will be transferred into the appropriate type of container. For small-scale extraction projects (less than 96 samples), low DNA yield (swabs) projects and for punctual genomic analysis projects, we usually transfer DNA into a regular half-skirt 96-well PCR plate. The plate is sealed with auto adhesive plastic film. For projects that require the creation of a DNA bank, and high-yield projects DNA projects (when recommended volume of blood, buffy coat, saliva, or recommended amounts of cells are sent), we transfer your samples into 1 ml individual microtubes with screw cap, organized in a rack formatted like a 96-well plate. Each tube is identified with a linear barcode on the side of the tube, and a 2D barcode under tube bottom. Take note that a specialized reader is required to read the 2D barcode on the tube, but linear barcodes are also displayed as a readable number on the side of each tube. For more information about this product please visit the manufacturer's website Thermo Fisher Scientific for product Matrix 2D Barcoded Tubes and Rack Only these two types of containers are considered. Any request to transfer the samples to another type of container could generate additional costs and may result in the inability to quantify DNA and determine its quality. For technical reasons, only DNA samples arranged in plate format can be analyzed. Samples transferred to individual tubes will not be analyzed (DNA quantification and quality of the DNA). DNA concentration analysis The DNA extraction service includes DNA concentration measurement by UV spectrophotometry at 260nm for all samples. This method gives an approximate value of the DNA concentration of your samples. It is important to understand that this method may overestimate the DNA concentration, because any free molecule containing an aromatic ring will create some absorbance at 260nm. We recommend adding a DNA concentration measurement by PicoGreen method (optional). This quantification method measures only double-stranded DNA in your samples and is not affected by the presence of contaminating molecules. Note that the PicoGreen assay is included by default as a preliminary step for the majority of sequencing or genotyping projects submitted to Génome Québec. DNA quality analysis We evaluate the quality of extracted genomic DNA by UV spectrophotometry at 260 and 280nm. The 260/280 ratio gives an idea of the quality of the DNA extracted and if it is contaminated with RNA or proteins. A sample with a ratio close to 1.8 is generally considered as pure DNA". 10

11 Sample identification and results files As soon as the extraction project is completed, an will be sent to the contact person via the address provided in the request form. Once the results are available on the Nanuq account, you can then access all information related to your samples and various extraction processes directly in the project in your Nanuq account. Following the extraction project, we will send by the Excel files containing all information related to the identification of your samples, the identification of containers and the location of your samples in their respective container (96-well plate Matrix or 2D Tube Rack). The files related to the analysis of the concentration and quality of DNA you will also be provided. For more information Client management office Frédérick Robidoux, B.Sc. Sharen Roland, B.Sc. Philippe Daoust, M.Sc. McGill University and Génome Québec Innovation Center 740 Docteur Penfield Avenue, room 7104 Montréal (Québec) Canada H3A 0G1 Phone : Fax : infoservices@genomequebec.com 11