hygiena KIT230046 GMO RR Soya Quantification Kit User Guide

hygiena KIT230046 GMO RR Soya Quantification Kit

Product Information

• Product Name: GMO RR Soya Quantification Kit
• Product Number: KIT230046
• Revision: A, December 2023
• For: In Vitro Use Only

Intended Use: This kit is designed for quantifying GMO RR Soya in food samples for testing purposes.

Specifications

• Kit Size: 2x 64 reactions

• Amplification: 50 cycles

• Fluorescence Detection Channels: FAM: GTS 40-3-2 and Soya
  (Le1)

• Probe Quencher: TAMRA

• No Passive Reference Dye Included

Program Setup

Before setting up the PCR reactions, program your real-time PCR instrument with the following settings:

• Channels:
  • FAM: GTS 40-3-2 and Soya (Le1)
• Amplification:
  • 4 minutes at initial denaturation temperature
  • 10 minutes at annealing/extension temperature
  • 15 seconds at denaturation temperature
  • 60 seconds at annealing/extension temperature
• Cycles: 50 cycles
• Fluorescence Detection: 1 cycle at the end

Preparation of Standard Curve

Before starting the PCR reactions, prepare a standard curve using the following dilution steps:

Preparation of PCR Mix

Follow these steps to prepare the PCR mixes:

1. Take appropriate precautions to prevent contamination, such as using filter tips and wearing gloves.
2. Thaw reagents and briefly spin vials before opening (do not vortex!).
3. Mix the reagents according to the instructions provided in the product manual.

PCR Reactions Setup

Follow these steps to set up the PCR reactions:

1. Prepare the PCR mixes as instructed above.

2. Add the PCR mix to the appropriate wells or tubes.

3. Add the samples and controls to the designated wells or tubes.
   For sample DNA, dilute it at least 1:10 in the Dilution Buffer (blue cap).

4. Seal the strips or plate accurately to prevent contamination.

5. Briefly spin the strips or plate in a suitable centrifuge.

6. Start the real-time PCR run according to the program setup described above.

FAQ

Q: How many reactions does the kit support?

A: The kit supports 2x 64 reactions, providing a total of 128 reactions.

Q: Can I use this kit for purposes other than food testing?

A: No, this kit is specifically designed for GMO RR Soya quantification in food samples and should not be used for other purposes.

Q: What should I do if my real-time PCR instrument requires a probe quencher and a passive reference dye specification?

A: This kit contains probes with TAMRA as the quencher and does not include a passive reference dye. Please consult your instrument’s manual or contact the manufacturer for instructions on specifying these parameters.

Q: Where can I find the complete product manual?

A: The entire product manual is available on our website. It is strongly recommended to read the manual before using the product.

Ready Reference Guide

Revision A, December 2023

Product No. KIT230046
Before starting, it is strongly recommended to read the entire product manual available on our website.

PROGRAM SETUP

Program your real-time PCR instrument before setting up the PCR reactions. Select the following channels:

FAM: GTS 40-3-2 and Soya (Le1)

For some real-time PCR instruments the probe quencher as well as the use of a passive reference dye must be specified. This kit contains probes with TAMRA as quencher and no passive reference dye.

PREPARATION OF STANDARD CURVE

Use the Calibrator DNA (purple cap) and Dilution Buffer (blue cap) to prepare dilutions according to the table below. For each dilution step, pipet 30 µL (60 µL for duplicates) of Dilution Buffer into a new reaction tube. Transfer 10 µL (20 µL for duplicates) from the preceding step to new dilution step. Mix well between pipetting steps. The prepared dilutions can be used for both standard curves, GMO gene and reference gene.
A typical experiment consists of 16 wells needed for standards and controls, plus 2 × n wells (n = number of food samples). Since a multiwell plate has 96 wells, 40 food samples can be analyzed during one PCR run if the GMO gene and the reference gene are analyzed in the same run. Some real-time PCR instruments provide the opportunity to import external standard curves generated in a previous run; then, 46 food samples can be analyzed during one PCR run.

PREPARATION OF THE PCR MIX

Take appropriate precautions to prevent contamination, e.g., by using fi lter tips and wearing gloves. Thaw reagents, mix (do not vortex!), and briefl y spin vials before opening.
For data interpretation and calculation please refer to the entire product manual.

1. PREPARE PCR MIXES
   GMO PCR mix: Add 18 µL of Master Mix (yellow cap), 1 µL of Enzyme Solution (red cap) and 1 µL of Dye Solution (black cap) for each reaction to a suitable tube.
   Reference PCR mix: Add 18 µL of Master Mix (green cap), 1 µL of Enzyme Solution (red cap) and 1 µL of Dye Solution (black cap) for each reaction to a suitable tube.
   (n samples + 2 controls + at least one additional reaction to cover pipetting loss). Mix carefully but thoroughly by pipetting up and down.

2. ADD PCR MIX
   Pipet 20 µL of prepared GMO and Reference PCR mix into respective strip or plate well.

3. ADD SAMPLES AND CONTROLS
   Sample DNA must be diluted at least 1:10 in the Dilution Buffer (blue cap).
   To each PCR mix (GMO and Reference), pipet 5 µL of samples, standards, Negative Control (colorless cap) or Control Template (purple cap) into respective wells.

4. SEAL
   Seal strips/plate accurately.

5. CENTRIFUGE
   Briefl y spin strips/plate in a suitable centrifuge.

6. START REAL-TIME PCR RUN
   Cycle samples as described above.

foodproof® GMO RR Soya Quantification Kit

KIT230046
Kit for 2x 64 reactions Store kit at -15 to -25 °C

For food testing purposes FOR IN VITRO USE ONLY Made in Germany

Hygiena® | Camarillo, CA 93012 USA | diagnostics.support@hygiena.com | www.hygiena.com

References

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