Thermo Fisher Scientific RapidFinder Direct Lysis Protocol User guide

Brand: Thermo Fisher Scientific

Model: RapidFinder Direct Lysis Protocol

Type: User guide

RapidFinder™ Direct Lysis Protocol

Publication Number 4485956 Revision A

■Overview ............................................................ 1

■Required materials .................................................... 2

■Prepare DNA by direct lysis of enriched cultures .......................... 2

Appendix A Sample enrichment ........................................... 3

■Sample enrichment .................................................... 3

Documentation and Support ............................................... 4

■Obtaining SDSs ....................................................... 4

■Obtaining Certificates of Analysis ....................................... 4

■Obtaining support ..................................................... 4

■Limited product warranty .............................................. 4

WARNING! Read the Safety Data Sheets (SDSs) and follow the handling in‐

structions. Wear appropriate protective eyewear, clothing, and gloves. Safety

Data Sheets (SDSs) are available from www.lifetechnologies.com/support.

Overview

The RapidFinder™ Direct Lysis Protocol is a simple, rapid method for preparation of

nucleic acid for PCR from enriched cultures of food and environmental samples. The

protocol is intended for use with cultures enriched for E. coli O157:H7, Salmonella spp.,

Listeria monocytogenes, or Listeria spp.

Enriched cultures are treated with Proteinase K in a specially formulated Lysis Buffer

at 55°C for 30 minutes, followed by heat inactivation of the Proteinase K at 95°C. The

procedure can be completed in about 45 minutes with minimal hands‐on time. The ly‐

sate is ready for use in downstream PCR using one of the following detection kits as

appropriate for the pathogen of interest.

Detection kit Catalog number

MicroSEQ®

E. coli

O157:H7 Detection Kit 4427409

MicroSEQ®

Salmonella

spp. Detection Kit 4403930

MicroSEQ®

Listeria monocytogenes

Detection Kit 4403874

MicroSEQ®

Listeria

spp. Detection Kit 4427410

USER BULLETIN

For testing of Food and Environmental samples only.

Required materials

Unless otherwise specified, all materials are available from Life Technologies.

Item Source

Lysis Buffer, FS (7 mL)[1] Cat. no. 4480724

Proteinase K, FS (1.25 mL)[1] Cat. no. 4480715

Nuclease-free Water, 50 mL Cat. no. AM9937

MicroAmp® Optical 96-Well Reaction Plates Cat. no. N8010560

ABgene® Adhesive PCR Plate Seals, PCR Foil Thermo Scientific AB-0626 [2]

Thermal cycler, 96-well format:

GeneAmp® PCR System 9700 or equivalent

Visit www.lifetechnologies.com

Multi-channel pipettor MLS[3]

Aerosol-resistant pipette tips MLS

[1] Sufficient for 100 reactions.

[2] Recommended because it is designed to avoid clogged pipette tips.

[3] MLS: major laboratory supplier.

Prepare DNA by direct lysis of enriched cultures

Before you begin: culture food or environmental samples for the pathogen of interest

as described in Appendix A, “Sample enrichment”.

1. Prepare sufficient Lysis Solution Mix for the number of reactions required, plus

10% overage.

Component Volume per reaction

Lysis Buffer, FS 63 µL

Proteinase K, FS 10 µL

Nuclease-free Water 127 µL

2. Distribute Lysis Solution Mix and enriched cultures to the appropriate wells in a

standard 96‐well PCR plate.

Component Volume per well

Enriched culture 5 µL

Lysis Solution Mix 200 µL

3. Seal the plate with an aluminum plate seal.

Ensure that the wells are sealed properly, to avoid evaporation and cross‐con‐

tamination.

RapidFinder™ Direct Lysis Protocol

Required materials

RapidFinder™ Direct Lysis Protocol

4. Invert the plate at least 5 times to mix the enriched culture with Lysis Solution

Mix.

5. Load the plate in a thermal cycler and run the following program:

Temperature Time

55°C 30 min

95°C 10 min

4°C ∞[1]

[1] Hold at 4°C until the plate is removed for use in PCR or for storage.

The lysate is ready for PCR using the appropriate MicroSEQ® detection kit for the

pathogen of interest.

• Use 30 µL of lysate per PCR assay.

• Pipet the lysate directly from the plate by puncturing the aluminum seal with the

pipette tip.

Note: If you use a pierceable aluminum foil seal other than the one recommen‐

ded, be sure to check for clogged tips when pipetting samples.

STOPPING POINT Store the sealed plate at 4°C for up to 72 hours, or store long‐term at

–20°C.

If the seal has been punctured, place another seal on top of the original seal before

storage. Do not remove the original seal from the plate, to avoid cross‐contamination.

Appendix A Sample enrichment

Sample enrichment

Enrich food and environmental samples according to the ISO standards for the patho‐

gen of interest, with noted modifications.

Pathogen ISO standard Modification

E. coli

O157:H7 ISO 16654 Recommended: use Buffered Peptone

Water in place of modified Tryptone

Soy Broth.

Salmonella

spp. ISO 6579 None.

Listeria monocytogenes

ISO 11290-1 Secondary enrichment for 24 hours.

Listeria

spp.

Appendix A Sample enrichment

Sample enrichment

RapidFinder™ Direct Lysis Protocol

Documentation and Support

Obtaining SDSs

Safety Data Sheets (SDSs) are available from www.lifetechnologies.com/support

Note: For the SDSs of chemicals not distributed by Life Technologies, contact the

chemical manufacturer.

Obtaining Certificates of Analysis

The Certificate of Analysis provides detailed quality control and product qualification

information for each product. Certificates of Analysis are available on our website. Go

to www.lifetechnologies.com/support and search for the Certificate of Analysis by

product lot number, which is printed on the box.

Obtaining support

For the latest services and support information for all locations, go to:

www.lifetechnologies.com/support

At the website, you can:

• Access worldwide telephone and fax numbers to contact Technical Support and

Sales facilities

• Search through frequently asked questions (FAQs)

• Submit a question directly to Technical Support ([email protected])

• Search for user documents, SDSs, vector maps and sequences, application notes,

formulations, handbooks, certificates of analysis, citations, and other product

support documents

• Obtain information about customer training

• Download software updates and patches

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth

in the Life Technologies' General Terms and Conditions of Sale found on Life Tech‐

nologies' website at www.lifetechnologies.com/termsandconditions. If you have any

questions, please contact Life Technologies at www.lifetechnologies.com/support.

Documentation and Support

Obtaining SDSs

RapidFinder™ Direct Lysis Protocol

For testing of Food and Environmental samples only.

The information in this guide is subject to change without notice.

DISCLAIMER

LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, IN-

CLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY

LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR

ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM

THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

TRADEMARKS

The trademarks mentioned herein are the property of Life Technologies Corporation and/or its affiliate(s) or their respective owners. ABgene® is a registered

trademark of Abgene, Ltd.

Headquarters

5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288

For support visit lifetechnologies.com/support or email [email protected]

lifetechnologies.com

21 May 2013

Thermo Fisher Scientific RapidFinder Direct Lysis Protocol User guide

Brand: Thermo Fisher Scientific

Model: RapidFinder Direct Lysis Protocol

Type: User guide

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Thermo Fisher Scientific RapidFinder Direct Lysis Protocol User guide

Thermo Fisher Scientific RapidFinder Direct Lysis Protocol User guide

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