hygiena KIT230044 GMO Bt176 Maize Quantification Kit User Guide

hygiena KIT230044 GMO Bt176 Maize Quantification Kit

Product Information

Specifications

• Product Name: GMO Bt176 Maize Quantification Kit
• Product Number: KIT230044
• Revision: A
• Date: December 2023
• For in vitro use only
• Kit for 2 x 64 reactions

Product Usage Instructions

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

• Channels: FAM (Bt176) and Maize (zSSllb)
• Cycling Conditions:
  • Step 1: 4 minutes
  • Step 2: 10 minutes
  • Step 3: 15 seconds
  • Step 4: 60 seconds*
• Cycle: 1 cycle
• Number of Cycles: 50 cycles
• • Fluorescence detection

Note: For some real-time PCR instruments, you may need to specify the probe quencher and use a passive reference dye. This kit contains probes with TAMRA as the quencher and does not require a passive reference dye.

Preparation of Standard Curve
Before starting the PCR reactions, prepare a standard curve using the following dilutions:

Note: For detailed data interpretation and calculation, refer to the entire product manual available on our website.

Preparation of the PCR Mix
Follow these steps to prepare the PCR mix:

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. For detailed instructions on mixing and reagent ratios, refer to the entire product manual.

Addition of PCR Mix and Samples
Follow these steps to add the PCR mix and samples:

1. Prepare the PCR mixes as instructed.
2. Add the PCR mix to the designated wells or tubes.
3. Dilute sample DNA at least 1:4 in the Dilution Buffer (blue cap).
4.  Add the diluted samples and appropriate controls to thedesignated wells or tubes.

Sealing and Centrifugation
Follow these steps to seal and centrifuge the samples:

1. Accurately seal the strips/plate to prevent contamination.
2. Briefly spin the strips/plate in a suitable centrifuge.

Start Real-Time PCR Run
Once the samples are prepared and centrifuged, start the real-time PCR run with the programmed cycling conditions and the specified number of cycles.

FAQ

• Q: Can this kit be used for food testing purposes?
  A: Yes, this kit is suitable for food testing purposes.

PROGRAM SETUP

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

• FAM: Bt176 and Maize (zSSllb).

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 a 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 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 filter tips and wearing gloves. Thaw reagents, mix (do not vortex!) and briefly spin vials before opening. For data interpretation and calculation, refer to the entire product manual.

1. PREPARE PCR MIXES

   • GMO PCR mix: Add 13 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 13 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 15 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 1:4 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
   Briefly spin strips/plate in a suitable centrifuge.

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

foodproof®
GMO Bt176 Maize
Quantification Kit

KIT230044
Kit for 2 x 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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