hygiena KIT230112 LP Yeast and Mold Quantification LyoKit User Guide

hygiena KIT230112 LP Yeast and Mold Quantification LyoKit

Product Information

Specifications:

• Product Name: Yeast and Mold Quantification LyoKit
• Revision: A, November 2023
• Product Numbers : KIT230112 (LP), KIT230113 (RP), KIT230114 (DP)
• PCR Kit for quantitative detection of yeast and mold using real-time PCR instruments
• Contains probes with a non-fluorescent dark quencher and no passive reference dye

Product Usage Instructions

Program Setup:

1. Program your real-time PCR instrument before setting up PCR reactions. Select channels: FAM (yeast and mold) and HEX (Internal Control).
2. Follow the cycle settings provided in the manual for fluorescence detection.

Preparation of Standard Curve:
Dilute samples following the dilution factor table provided in the manual.

Preparation of the PCR Mix:

1. Take precautions to prevent contamination by using filter tips and gloves.
2. Place PCR tube strips in a rack after removing from the aluminum bag.
3. Decap tubes just before filling, discard caps immediately.
4. Add samples and controls to the tubes.
5. Seal the tubes with provided 8-cap strips securely.
6. Mix thoroughly to resuspend the pellet.
7. Centrifuge briefly to ensure proper mixing.
8. Start the real-time PCR run following the cycling instructions.

FAQ

• Q: Can this kit be used for food testing?
  A: Yes, this kit can be used for food testing purposes. It is for in vitro use only.

• Q: Where is the product manufactured?
  A: This product is made in Germany.

foodproof®
Yeast and Mold Quantification LyoKit

Ready Reference Guide

• Revision A, November 2023
• Product No. KIT230112 (LP), KIT230113 (RP), KIT230114 (DP)
• PCR kit for the quantitative detection of yeast and mold using real-time PCR instruments.
• 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 (yeast and mold) and HEX (Internal Control).

• Pre-incubation: 1 cycle
  • Step 1: 37 °C for 5 min
  • Step 2: 95 °C for 10 min
• Amplification 50: cycles
  • Step 1 : 95 °C for 5 sec
  • Step 2*: 63 °C for 60 sec
  • Step 3 : 72 °C for 60 sec
  • • Fluorescence detection

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 a non- fluorescent “dark” quencher and no passive reference dye.

PREPARATION OF STANDARD CURVE

• Use the Quantification Standard (vial 2, purple cap) and Negative Control (vial 3, colorless cap) to prepare dilutions according to the table below.
• For each dilution step, pipet 90 µL of Negative Control into a new reaction tube. Transfer 10 µL from preceding step to 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 (n = number of food samples).

Dilution Step| Dilution Factor| Concentration to Be Entered as Standard (GE/reaction)
---|---|---
1| Undiluted| 10,000
2| 1:10| 1,000
3| 1:100| 100
4| 1:1,000| 10

PREPARATION OF THE PCR MIX

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

1. PLACE STRIPS IN RACK
   Take needed number of PCR tube strips out of aluminum bag.
   Important: close bag tightly afterwards. 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.

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

3. 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.

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

5. MIX
   Resuspend pellet by mixing thoroughly.

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

7. 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®

• Yeast and Mold Quantification LyoKit
• KIT230112 / 13 / 14 Kit for 96 reactions Store kit at 2 to 8 °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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