PCRmax Genesig NOS terminator in GM crops Instruction Manual

NOS terminator

NOS terminator (GMO)

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

1

Quantification of NOS terminator (GMO) genomes.

Advanced kit handbook HB10.07.08

Published Date: 26/04/2016

The kit provides a method for detecting gene insertion events by real-time PCR. The kit is
based on the PCR amplification and detection of the Agrobacterium tumefaciens
(A.tumefaciens) NOS terminator. This sequence has been widely used as a transcriptional
terminator in synthetic gene constructs.

Since A.tumefaciens is naturally found in the soil, it is necessary to exclude false positive
results by proving that the wild type virus has not infected or contaminated a non GM
sample. This kit provides a control test that detects a region of the A.tumefaciens genome
that is not used in the genetic modification of plants.

A positive signal with the AT-tNOS-GM test indicates the presence of GM material in the
sample, provided that no signal is obtained from the AT-tNOS-WT test.

Where signals are obtained with the AT-tNOS-WT test this indicates that naturally
occurring A.tumefaciens is present in the samples. The data must be carefully considered
by looking at the relative signal strength between the AT-tNOS-WT test and the AT-tNOS­GM test in the samples.
Within the details result view you can review the Cq values for each of the tests. If the AT­tNOS-GM test detection occurs 5 or more Cq values earlier than the AT-tNOS-WT control
test then the test confirms the presence of both the wild A.tumefaciens organism and GM
plant material. If the signal strength for the AT-tNOS-WT and AT-tNOS-GM are closer
together with a smaller difference in Cq values then the test is indeterminate since the
presence of wild A.tumefaciens prevents the analysis of the GM content of the sample.

Introduction to NOS terminator (GMO)

2

Quantification of NOS terminator (GMO) genomes.

Advanced kit handbook HB10.07.08

Published Date: 26/04/2016

MAX

MIN

This kit is designed to have 100% homology with all available sequence data on the
Agrobacterium tumefaciens (A.tumefaciens) NOS terminator.
The control test detects a region of the A.tumefaciens genome that is not used in the
genetic modification of plants.

If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to [e-mail] and our bioinformatics team will
answer your question.

Specificity

3

Quantification of NOS terminator (GMO) genomes.

Advanced kit handbook HB10.07.08

Published Date: 26/04/2016

Kit Contents

• A. tumefaciens NOS terminator primer/probe mix (150 reactions BROWN)

FAM labelled

• A. tumefaciens bacterium primer/probe mix (150 reactions BROWN)

FAM labelled

• A. tumefaciens NOS terminator positive control template (for Standard curve RED)

• A. tumefaciens bacterium positive control template (for Standard curve RED)

• Internal extraction control primer/probe mix (150 reactions BROWN)

VIC labelled as standard

• Internal extraction control DNA (150 reactions BLUE)

• Endogenous control primer/probe mix (150 reactions BROWN)

FAM labelled

• RNAse/DNAse free water (WHITE)

for resuspension of primer/probe mixes and internal extraction control DNA

• Template preparation buffer (YELLOW)

for resuspension of positive control templates and standard curve preparation

Reagents and equipment to be supplied by the user

Real-Time PCR Instrument

DNA extraction kit

This kit is designed to work well with all processes that yield high quality DNA with minimal
PCR inhibitors.

Lyophilised 2x qPCR Mastermix

This kit is designed to be compatible with all commercially available Mastermixes that run
with standard cycling conditions.

Pipettors and Tips

Vortex and centrifuge

Thin walled 1.5 ml PCR reaction tubes

4

Quantification of NOS terminator (GMO) genomes.

Advanced kit handbook HB10.07.08

Published Date: 26/04/2016