hygiena KIT230230, KIT230231 foodproof Clostridium Quantification LyoKit User Guide

: August 16, 2024
: Hygiena

Table of Contents

hygiene KIT230230, KIT230231 foodproof Clostridium Quantification LyoKit
PROGRAM SETUP
PREPARATION OF A STANDARD CURVE
PREPARATION OF THE PCR MIX
References
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hygiene KIT230230, KIT230231 foodproof Clostridium Quantification LyoKit

Product No. KIT230230 (LP), KIT230231(RP) PCR kit for the quantitative detection of Clostridium spp. using real-time PCR instruments. Before starting, it is strongly recommended to read the entire product instructions available on our website.

PROGRAM SETUP

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

FAM (Clostridium spp.) and HEX (Internal Control).

For some real-time PCR instruments, the probe quencher as well as the usage of a passive reference dye has to be specified. This kit contains probes with a non-fluorescent “dark” quencher and no passive reference dye.

PREPARATION OF A STANDARD CURVE

Use the Quantification Standard (vial 2, purple cap) and Negative Control (vial 3, colorless cap) to prepare serial dilutions according to the table below. For each dilution step, pipet 90 µL Negative Control into a new reaction tube. Transfer 10 µL from the preceding step to the new dilution step. Mix well between pipetting steps. A typical experiment consists of 9 wells needed for standards (duplicates) and negative control, plus n wells for samples (n = number of food samples).

Dilution Step| Dilution Factor| Concentration to be entered as Standard (GU/reaction)
---|---|---
1| Undiluted| 100,000
2| 1:10| 10,000
3| 1:100| 1,000
4| 1:1,000| 100

foodproof® is a registered trademark of Hygiena. Any above-mentioned real-time PCR instruments are registered trademarks of their respective holders.

PREPARATION OF THE PCR MIX

Take appropriate precautions to prevent contamination, e.g., by using aerosol- barrier tips and wearing gloves. For data interpretation and calculation, refer to the complete product instructions.

PLACE STRIPS IN THE RACK
Take the needed number of PCR tube strips out of an aluminum bag. Important: close the bag tightly afterward. Place strips in a suitable PCR tube rack. If needed, gently tap the tubes to move the lyophilized pellets to the bottom of each tube.

UNCAP
Immediately before filling, open strips carefully and discard caps. Do not leave it open longer than necessary.

ADD SAMPLES AND CONTROLS
Pipet 25 µL of samples, standards, and Negative Control (colorless cap) into respective wells. If using less volume for samples, add Negative Control to reach 25 µL.

SEAL
Seal the tubes with the provided 8-cap strips properly.

MIX
Resuspend the pellet by mixing thoroughly.

CENTRIFUGE
Briefly spin strips, e.g., 5 seconds at 500 – 1,000 x g in a suitable centrifuge.

START REAL-TIME PCR RUN
Cycle samples as described above. Place tubes in a vertical, balanced order into the cycler, e.g., two strips can be placed in the first and last columns.

foodproof® Clostridium Quantification LyoKit KIT230230/ 31 Kit for 96 reactions Store kit at 2 to 8 °C For food testing purposes FOR IN VITRO USE ONLY Made in Germany