hygiena KIT230186 StarPrep Two 8-Strip Kit Instruction Manual

hygiena KIT230186 StarPrep Two 8-Strip Kit

Specifications

• Product Name: StarPrep Two 8-Strip Kit Aspergillus
• Product Number: KIT230186
• Kit Size: 480 reactions
• Revision: A, April 2024

Product Information

Overview

The StarPrep Two 8-Strip Kit is designed for high-throughput extraction of DNA from Aspergillus for direct use in PCR. It contains all necessary components for efficient DNA extraction.

Kit Contents

The kit includes:

1. 5 x 96 micro tube rack with 8-tube strips 1.2 mL (prefilled
   with beads) and 8-cap strips

2. 5 bottles with 30 mL lysis buffer

3. 5 bags with 12 x 8-cap strips

4. Quick Guide

Product Usage Instructions

Required Materials

Most required equipment and reagents are available through Hygiena Diagnostics. Please contact for further information. Ensure to use the materials listed below for optimal performance:

• Reagent D (Product No. KIT230001) – Only for Procedure C (2.3.3)
• Sterile reservoir (25 mL or 100 mL)
• 8-strip tubes, 1.2 mL – 12 x (96 well)

Precautions and Preparations

Before starting the extraction process, ensure all materials are at the correct temperature and follow proper laboratory safetyprotocols.

Workflows

Extraction Procedure A: Standard

Detailed steps for standard DNA extraction procedure.

Extraction Procedure B: High Purity

Detailed steps for high purity DNA extraction procedure.

Extraction Procedure C: Live/Dead

Detailed steps for live/dead DNA extraction procedure using Reagent D.

Troubleshooting

If you encounter any issues during the extraction process, refer to the troubleshooting section in the manual for guidance.

Support

If you need further assistance or have any questions, contact our support team for help.

FAQ

• How many reactions is the kit designed for?
  • The kit is designed for 480 reactions.
• Where can I get additional reagents and equipment?
  • Most required equipment and reagents are available through Hygiena Diagnostics. Contact them for further information.

OVERVIEW

The foodproof® StarPrep® Two 8-Strip Kit is designed for the rapid preparation of DNA from yeast or mold like Aspergillus for direct use in PCR. Up to 96 samples can be processed in parallel. In less than 30 minutes, the kit generates PCR template DNA from up to 1,000 μL of enrichment culture. The obtained DNA can be used directly in any PCR application. The special lysis buffer eliminates the need for hazardous organic extractions or chaotropic agents. The entire DNA preparation can be performed in a single tube, minimizing handling steps and exposure to biohazardous material. The reduced number of handling steps results in time saving and, because transfer steps of DNA-containing extracts are not necessary, the cross-contamination risks are minimized.

General Information

Number of Reactions The kit is designed for 480 reactions. Storage Conditions Store at 15 to 25 °C. The components of the foodproof StarPrep Two 8-Strip Kit are guaranteed to be stable through the expiration date printed on the label.

Applicability

The lysis buffer can be used to prepare DNA from up to 1,000 μL sample. The lysis buffer is optimized for the preparation of various types of sample material. The quality of the DNA obtained with the lysis buffer is suitable for any PCR application

Kit Contents

A schematic representation of the foodproof StarPrep Two 8-Strip Kit with all components:

1. 5 x 96 micro tube rack with 8-tube strips 1.2 mL (prefilled with beads) and 8-cap strips
2. 5 bottles with 30 mL lysis buffer
3. 5 bags with 12 x 8-cap strips
4. Quick Guide

INSTRUCTIONS

This section provides all information for a straightfoward DNA extraction from a variety of different food matrices.

Required Material

Most of the required equipment and reagents are available through Hygiena Diagnostics. Please contact us for further information.

It is highly recommended to only use the materials described below to ensure the performance of the method.

Precautions and Preparations

Follow all universal safety precautions governing work with biohazardous materials, e.g., wear lab coats and gloves at all times. Properly dispose of all contaminated materials, decontaminate work surfaces, and use a biosafety cabinet whenever aerosols might be generated. For more information, please refer to the appropriate material safety data sheet (SDS). The SDS is available online at www.hygiena.com.

Workflows

• Chapter 2.3 provides workflows for a qualitative analysis of molds like Aspergillus in different cannabis or hemp matrices.
• The Standard protocol (2.3.1) describes the DNA extraction from up to 1,000 μL enrichment culture.
• The High Purity protocol (2.3.2) describes the DNA extraction from difficult matrices. Inhibitory effects of the matrix are reduced by an additional wash step. The Live/Dead protocol (2.3.3) describes the DNA extraction including a step for live and dead cell differentiation with Reagent D.

EXTRACTION PROCEDURE A: STANDARD

The following protocol describes the DNA isolation from 1,000 μL enrichment culture using 8-tubes strips and multichannel pipettes. This short protocol is recommended for matrices like plant flowers and oils.

1. SHAKE SAMPLE
   Shake enrichment culture gently and let suspension settle for 5 to 10 min.

2. REMOVE CAPS
   Remove the 8-cap strips from the 8-tube strips. To minimize the contamination risk, use the decapper 8-strip tool.

3. ADD SAMPLE
   Transfer 1,000 µL sample (enrichment culture supernatant) to the 8-tube strips.

4. SEAL TUBES
   Seal the tubes with sterile cap strips.

5. CENTRIFUGE
   10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.
   Note: Time and g-force depend on the centrifuge (please see 2.1 Required Material for more information). Set the centrifuge acceleration to maximum speed and the brake to medium. If necessary, centrifugation forces should be calculated according to the manual for the centrifuge used.

6. REMOVE CAPS
   Remove and discard the 8-cap strips from the 8-tube strips. To minimize the contamination risk, use the decapper 8-strip tool.

7. REMOVE SUPERNATANT
   Remove only 900 µL supernatant carefully with a multichannel pipettor immediately after centrifugation, discard and inactivate appropriately. 100 µL has to remain in the tube. Take care that the pipette tips are not touching the pellets in the reaction tubes.

8. PREPARE LYSIS BUFFER
   Transfer required lysis buffer to a sterile reservoir. Prepare 300 µL lysis buffer per sample plus 1 mL lysis buffer as dead volume.
   Note: Shake the bottle with lysis buffer gently immediately before pipetting to avoid sedimentation of ingredients.

9. ADD LYSIS BUFFER AND MIX
   Pipet lysis buffer up and down 5 to 10 times in reservoir before using it to avoid sedimentation of ingredients.
   Transfer 300 µL lysis buffer with a multichannel pipettor to each tube. Resuspend pellets by pipetting up and down 5 to 10 times.
   Note: For optimal DNA isolation efficiency, pellet has to be completely resuspended.

10. SEAL TUBES
    Seal the tubes tightly with new sterile cap strips.

11. INSTALL ADAPTER SET
    Place the rack without rack lid in the TissueLyser Adapter Set.
    Note: Split the tube strips into 2 groups and place them into two tube racks to prepare appropriate counterweights for disruption. Place the tube strips into the racks in an even and balanced manner. Make sure each rack contains at least two strips placed in the outermost positions (rows 1 and 12 of the rack). If you prepare only a few samples (less than 4 strips) it is necessary to use additional tube strips prefilled with 400 µL water to balance the weight evenly.

12. MECHANICAL DISRUPTION
    Place the 2 adapters in the cell disruption unit and run mechanical disruption Mixer Mill 400: 15 min at 30 Hz. The efficiency of disruption depends on the mechanical cell disruption unit.

13. INCUBATE
    Remove tube rack bottom and install incubation frame. Incubate rack with tube stripes 5 min at 100 °C in TH 21 Heating Block for 8-tube strips. Weigh caps down with the lid weight.
    Note: To avoid removing and reinstalling the bottom, it is possible to place tube strips in an empty micro tube rack (with rack bottom removed).

14. CHILL
    Carefully remove the rack with the tube strips together with the lid weight from the heating unit and let it cool 3 – 5 min at room temperature. To avoid opening of caps, do not remove the lid weight until the strips have cooled down.

15. CENTRIFUGE
    Reinstall tube rack bottom. Centrifuge 10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.
    SUPERNATANT FOR DETECTION
    Use 5 µL supernatant in combination with foodproof PCR Kits.
    Note: Strictly avoid transferring fractions of the sediment to the PCR reaction because this might cause PCR inhibition.
    For later analysis, store DNA at -15 to -25 °C. After thawing, mix briefly by vortexing and centrifuge at 2,000 × g for 10 min. Note: The sample is not purified. Proteins, RNA, and other materials remain in the sample. Long-term storage or archival of prepared DNA samples is not recommended.

EXTRACTION PROCEDURE B: HIGH PURITY

This protocol includes a wash step. As a result it reduces inhibitory effects of the used matrix or enrichment culture media. This protocol is recommended for matrices like edibles and non-edibles.

1. SHAKE SAMPLE
   Shake enrichment culture gently and let suspension settle for 5 to 10 min.

2. REMOVE CAPS
   Remove the 8-cap strips from the 8-tube strips and keep sterile for later use. To minimize the contamination risk, use the decapper 8-strip tool.

3. ADD SAMPLE
   Transfer 1,000 µL sample (enrichment culture supernatant) to the 8-tube strips.

4. SEAL TUBES
   Seal the tubes with sterile cap strips.

5. CENTRIFUGE
   10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.
   Note: Time and g-force depend on the centrifuge (please see 2.1 Required Material for more information). Set the centrifuge acceleration to maximum speed and the brake to medium. If necessary, centrifugation forces should be calculated according to the manual for the centrifuge used.

6. REMOVE CAPS
   Remove and discard the 8-cap strips from the 8-tube strips. To minimize the contamination risk, use the decapper 8-strip tool.

7. REMOVE SUPERNATANT
   Remove only 900 µL supernatant carefully with a multichannel pipettor immediately after centrifugation, discard and inactivate appropriately. 100 µL has to remain in the tube. Take care that the pipette tips are not touching the pellets in the reaction tubes.

8. ADD WASH BUFFER
   Add 600 µL sterile double-distilled water to wash the pellet.

9. MIX
   Resuspend pellets by pipetting up and down 5 to 10 times.
   Note: For optimal DNA isolation efficiency, pellet has to be completely resuspended.

10. SEAL TUBES
    Seal the tubes tightly with new sterile cap strips.
    Note: Use additional consumable: 8-cap strips for micro tube rack, see 2.1 Material Required.

11. CENTRIFUGE
    10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.

12. REMOVE CAPS
    Remove and discard the 8-cap strips from the 8-tube strips.To minimize the contamination risk, use the decapper 8-strip tool.

13. REMOVE SUPERNATANT
    Remove only 600 µL supernatant carefully with a multichannel pipettor immediately after centrifugation, discard and inactivate appropriately. 100 µL has to remain in the tube. Take care that the pipette tips are not touching the pellets in the reaction tubes.

14. PREPARE LYSIS BUFFER
    Transfer required lysis buffer to a sterile reservoir. Prepare 300 µL lysis buffer per sample plus 1 mL lysis buffer as dead volume.
    Note: Shake the bottle with lysis buffer gently immediately before pipetting to avoid sedimentation of ingredients.

15. ADD LYSIS BUFFER AND MIX
    Pipet lysis buffer up and down 5 to 10 times in reservoir before using it to avoid sedimentation of ingredients. Transfer 300 µL lysis buffer with a multichannel pipettor to each tube. Resuspend pellets by pipetting up and down 5 to 10 times.
    Note: For optimal DNA isolation efficiency, pellet has to be completely resuspended.

16. SEAL TUBES
    Seal the tubes tightly with new sterile cap strips.

17. INSTALL ADAPTER SET
    Place the rack without rack lid in the TissueLyser Adapter Set.
    Note: Split the tube strips into 2 groups and place them into two tube racks to prepare appropriate counterweights for disruption. Place the tube strips into the racks in an even and balanced manner. Make sure each rack contains at least two strips placed in the outermost positions (rows 1 and 12 of the rack). If you prepare only a few samples (less than 4 strips) it is necessary to use additional tube strips prefilled with 400 µL water to balance the weight.

18. MECHANICAL DISRUPTION
    Place the two adapters in the cell disruption unit and run mechanical disruption Mixer Mill 400: 15 min at 30 Hz. The efficiency of disruption depends on the mechanical cell disruption unit.

19. INCUBATE
    Remove tube rack bottom and install incubation frame. Incubate rack with tube stripes 5 min at 100 °C in TH 21 Heating Block for 8-tube strips. Weigh caps down with the lid weight.
    Note: To avoid removing and reinstalling the bottom, it is possible to place tube strips in an empty micro tube rack (with rack bottom removed).

20. CHILL
    Carefully remove the rack with the tube strips together with the lid weight from the heating unit and let it cool 3 – 5 min at room temperature. To avoid opening of caps, do not remove the lid weight until the strips have cooled down.

21. CENTRIFUGE
    Reinstall tube rack bottom. Centrifuge 10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.
    SUPERNATANT FOR DETECTION
    Use 5 µL supernatant in combination with foodproof PCR Kits.
    Note: Strictly avoid transferring fractions of the sediment to the PCR reaction because this might cause PCR inhibition. For later analysis, store DNA at -15 to -25 °C. After thawing, mix briefly by vortexing and centrifuge at 2,000 × g for 10 min. Note: The sample is not purified. Proteins, RNA, and other materials remain in the sample. Long-term storage or archival of prepared DNA samples is not recommended.

EXTRACTION PROCEDURE C: LIVE/DEAD

The following protocol describes the DNA isolation including a step for live and dead cell differentiation with Reagent D.

1. SHAKE SAMPLE
   Shake enrichment culture gently and let suspension settle for 5 to 10 min.

2. ADD SAMPLE (RACK 1)
   Transfer 1,000 µL sample (enrichment culture supernatant) to the empty 8-Tube strips (without beads) of rack 1.
   Note: Use additional consumable: 96 MicroTube rack with 8-tube strips 1.2 mL, see 2.1 Material Required.

3. SEAL TUBES
   Seal the tubes of rack 1 tightly with new sterile cap strips.
   Note: Use additional consumable: 8-cap strips for micro tube rack, see 2.1 Material Required.

4. CENTRIFUGE
   10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.
   Note: Time and g-force depend on the centrifuge (please see 2.1 Required Material for more information). Set the centrifuge acceleration to maximum speed and the brake to medium. If necessary, centrifugation forces should be calculated according to the manual for the centrifuge used.

5. REMOVE CAPS
   Remove and discard the 8-cap strips from the 8-tube strips. To minimize the contamination risk, use the decapper 8-strip tool.

6. REMOVE SUPERNATANT
   Remove only 900 µL supernatant carefully with a multichannel pipettor immediately after centrifugation, discard and inactivate appropriately. Take care that the pipette tips are not touching the pellets in the reaction tubes.

7. PREPARE REAGENT D
   Transfer an adequate volume of Reagent D in a sterile reservoir: 300 µL per sample and 1 mL as dead volume.
   Note: The lights in the clean bench must be switched off. Proceed immediately with the following steps of the protocol. Avoid extended exposure to light.

8. ADD REAGENT D AND MIX
   Transfer 300 µL Reagent D with a multichannel pipettor to each tube of rack 1. Resuspend pellets by pipetting up and down 5 times.
   Note: For optimal DNA isolation efficiency, pellet has to be completely resuspended. For uptake of Reagent D and mix, pipet with maximum speed of the automatic pipette. Proceed immediately with the following steps of the protocol. Avoid extended exposure to light.

9. D-LIGHT TREATMENT
   Do not seal the 8-tube strips with with 8-cap strips. Close the 8-tube strips of rack 1 with the transparent rack lid only and place it in the D-Light unit. Incubate first in the dark for 10 min and subsequently expose to light for 5 min at room temperature in the D-Light unit.

10. REMOVE CAPS (RACK 2)
    Remove and discard the 8-cap strips from a new 8-tube strip with beads (Rack 2). To minimize the contamination risk, use the decapper 8-strip tool.

11. ADD VOLUMES OF RACK 1 TO RACK 2
    Resuspend samples in rack 1 five times. Transfer 400 µL volume from the 8-tube strips of rack 1 (from step 9) with a multichannel pipettor to the 8-tube strips with beads and wash buffer of rack 2. Mix five times.
    Note: For uptake and mix pipet with maximum speed of the automatic pipettor .

12. SEAL TUBES
    Seal the tubes of rack 2 tightly with sterile cap strips.

13. CENTRIFUGE
    Reinstall tube rack bottom.
    Centrifuge 10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.

14. REMOVE CAPS
    Remove and discard the 8-cap strips from the 8-tube strips. To minimize the contamination risk, use the decapper 8-strip tool.

15. REMOVE SUPERNATANT
    Remove 300 µL supernatant carefully with a multichannel pipettor immediately after centrifugation, discard and inactivate appropriately. Take care that the tips of the pipette in the reaction tubes are not touching the pellets.

16. PREPARE LYSIS BUFFER
    Transfer required lysis buffer to a sterile reservoir. Prepare 300 µL lysis buffer per sample plus 1 mL lysis buffer as dead volume.
    Note: Shake the bottle with lysis buffer immediately before pipetting to avoid sedimentation of ingredients.

17. ADD LYSIS BUFFER AND MIX
    Pipet lysis buffer up and down 5 to 10 times in reservoir before using it to avoid sedimentation of ingredients. Transfer 300 µL lysis buffer with a multichannel pipettor to each tube. Resuspend pellets by pipetting up and down 5 to 10 times.
    Note: For optimal DNA isolation efficiency, pellet has to be completely resuspended.

18. SEAL TUBES
    Seal the tubes tightly with new sterile cap strips.

19. INSTALL ADAPTER SET
    Place the rack without rack lid in the TissueLyser Adapter Set.
    Note: Split the tube strips into 2 groups and place them into two tube racks to prepare appropriate counterweights for disruption. Place the tube strips into the racks in an even and balanced manner. Make sure each rack contains at least two strips placed in the outermost positions (rows 1 and 12 of the rack). If you prepare only a few samples (less than 4 strips) it is necessary to use additional tube strips prefilled with 400 µL water to balance the weight.

20. MECHANICAL DISRUPTION
    Place the 2 adapters in the cell disruption unit and run mechanical disruption Mixer Mill 400: 15 min at 30 Hz. The efficiency of disruption depends on the mechanical cell disruption unit.

21. INCUBATE
    Remove tube rack bottom and install incubation frame. Incubate rack with tube stripes 5 min at 100 °C in TH 21 Heating Block for 8-tube strips. Weigh caps down with the lid weight.
    Note: To avoid removing and reinstalling the bottom, it is possible to place tube strips in an empty micro tube rack (with rack bottom removed).

22. CHILL
    Carefully remove the rack with the tube strips together with the lid weight from the heating unit and let it cool 3 – 5 min at room temperature. To avoid opening of caps, do not remove the lid weight until the strips have cooled down.

23. CENTRIFUGE
    Reinstall tube rack bottom. Centrifuge 10 min at 5,400 x g (or 25 min at 2,000 x g). Make sure the rack is not sealed with rack lid during centrifugation.

SUPERNATANT FOR DETECTION

Use 5 µL supernatant in combination with foodproof PCR Kits.

Note: Strictly avoid transferring fractions of the sediment to the PCR reaction because this might cause PCR inhibition. For later analysis, store DNA at -15 to -25 °C. After thawing, mix briefly by vortexing and centrifuge at 2,000 × g for 10 min. Note: The sample is not purified. Proteins, RNA, and other materials remain in the sample. Long-term storage or archival of prepared DNA samples is not recommended.

Troubleshooting

DNA extract, e.g., 1:5 or reduce the amount of extracted DNA.
Some of the centrifugation pellet transferred over to the PCR.| Always centrifuge the DNA sample before performing PCR.

Do not allow the filter tip to have contact with the pellet.

Supernatants are not completely removed.| Remove supernatants completely (e.g., after Reagent D treatment).
Low DNA yield.| Improper storage of kit components.| Store kit reagents at 15 to 25 °C.
Sample contains substances that reduce the DNA extraction efficiency.| Reduce the sample volume.
Pellet resuspension incomplete.| Improve resuspension by prolonged pipetting or vortexing.
Suboptimal reaction conditions.| Ensure proper heating conditions.

Verify correct temperature of the heating block with a thermometer.

Lid of the reaction tube opens during or after heating.| Reaction tube not firmly closed or not enough weight exerted on the caps of the tube strips.| Ensure that all reaction tubes are firmly closed before heating.

Weigh the caps down during heating and do not remove the weight until the tubes have cooled down.

Support

If you have questions or experience any problems with our products, please contact us: www.hygiena.com/support Our aim is to provide you with a solution as quickly and effectively as possible. We would also like you to contact us if you have any suggestions for improving the product or in case you would like to use our product for a different application. We highly value your feedback.

ADDITIONAL INFORMATION

Quality Control

All products are regularly monitored by our quality control. You can find the certificate of analysis (COA) on our website. If you would like to carry out your own quality control, you will find the analysis method described in the certificate. Waste Disposal All contaminated and potentially infectious material, like enrichment cultures or food samples, should be autoclaved before disposal and eliminated according to local rules and regulations. For proper disposal of unused chemicals, please refer to the SDS. Warranty and Disclaimer of Liability “Limited Warranty” and “Disclaimer of Liability”: Hygiena Diagnostics GmbH warrants that this product is free from defects in materials and workmanship through the expiration date printed on the label and only if the following are complied with:

1. The product is used according to the guidelines and instructions set forth in the product literature;
2. Hygiena Diagnostics GmbH does not warrant its product against any and all defects when: the defect is as a result of material or workmanship not provided by Hygiena Diagnostics GmbH; defects caused by misuse or use contrary to the instructions supplied, or improper storage or handling of the product;
3. All warranties of merchantability and fitness for a particular purpose, written, oral, expressed or implied, shall extend only for a period of one year from the date of manufacture. There are no other warranties that extend beyond those described on the face of this warranty;
4. Hygiena Diagnostics GmbH does not undertake responsibility to any purchaser of its product for any undertaking, representation or warranty made by any dealers or distributors selling its products beyond those herein expressly expressed unless expressed in writing by an officer of Hygiena Diagnostics GmbH;
5. Hygiena Diagnostics GmbH does not assume responsibility for incidental or consequential damages, including, but not limited to responsibility for loss of use of this product, removal or replacement labor, loss of time, inconvenience, expense for telephone calls, shipping expenses, loss or damage to property or loss of revenue, personal injuries or wrongful death;
6. Hygiena Diagnostics GmbH reserves the right to replace or allow credit for any modules returned under this warranty.

Trademarks

foodproof, microproof®, vetproof® , ShortPrep® , RoboPrep® and LyoKit® are trademarks of Hygiena Diagnostics GmbH. Other brand or product names are trademarks of their respective holders.

Reference Number

The reference number and original Hygiena Diagnostics GmbH article number: S 400 17 L

Change Index

Version 1, April 2021: New document layout and content. Revision A, April 2024: Rebranding and new layout; updated product numbers. S 400 17 L 20 -> INS-KIT230186-2-REVA

Hygiena®

• Camarillo, CA 93012
• USA
• diagnostics.support@hygiena.com

Manufactured by

• Hygiena Diagnostics GmbH
• Hermannswerder 17
• 14473 Potsdam
• Germany
• www.hygiena.com

References

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