hygiena StarPrep Two 8 Strip High Throughput Kit Instruction Manual

hygiena StarPrep Two 8 Strip High Throughput Kit

OVERVIEW

The foodproof® StarPrep Two 8-Strip Kit is designed for the rapid preparation of DNA from gram-positive bacteria like Listeria monocytogenes for direct use in PCR. Up to 96 samples can be processed in parallel. The kit generates PCR template DNA from up to 800 μl of enrichment culture. The extracted DNA can be used directly in any PCR application.

The StarPrep Two 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 hazardous material. The reduced number of handling steps saves time and eliminating DNA- containing extract transfer steps minimizes the risk of cross-contamination.

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 800 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:

KIT 2301 86:

1. 5x 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 12x 8-cap strips

INSTRUCTIONS

This section provides all information for a seamless DNA extraction from a variety of matrices.

Required Material
Most of the required equipment and reagents are available through HygienaTM.
Please contact us for further information.

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

Consumables

• Sterile reservoir, 100 ml
  for procedures A.1: STANDARD (2.4.1) and B: RAPID (2.4.3)

• Automation friendly reservoirs
  sterile 150 ml, reservoir base and lid – INTEGRA Biosciences
  only for procedure A.2: VIAFLO96 (2.4.2)

Equipment

• Multichannel pipette and filter tips
  e.g., 8-Channel Pipette VIAFLO – INTEGRA Biosciences); GripTips: 50 to 1,250 μl
  or EP Xplorer Plus Electronic Multichannel Pipette; Filter Tips: 50 to 1,250 μl
  for procedures A.1: STANDARD (2.4.1) and B: RAPID (2.4.3)

• Benchtop pipetting system and deep well tips
  VIAFLO 96 base unit, 96 channel pipetting head,spring-loaded plate holder A & B for 96 well plates – all INTEGRA Biosciences; GripTips in racks: 50 to 1,250 μl
  only for procedure A.2: VIAFLO96 (2.4.2)

• Unit for mechanical cell disruption suitable for working with 1.2 ml x 8-tube strips MPS-1 High-Speed Multi Plate Shaker BioSan and 96 x 2 ml deepwell plate
  or Mixer Mill 400 Retsch GmbH with rack adapter TissueLyser Adapter Set 2×96 Qiagen

• Centrifuge with swing-out rotor for microtiter plates capable
  of a > 5,400 × g centrifugal force
  e.g., Sigma 4-16S including rotor
  Or centrifuge with swing-out rotor for microtiter plates capable
  of a 2,000 × g centrifugal force
  e.g., Sigma 2-7 including rotor

• TH 21 heating block thermostat

• Exchange block for deepwell plates for TH 21

• Lid weight with incubation frame for TH 21 heating block thermostat

• Decapper 8-strip

Recommended:

• Cap installing tool

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.bc- diagnostics.com.

• Always use filter tips in order to avoid cross-contamination.

• To reach the required temperature of 95 – 100 °C in the tubes for the lysis step of the bacteria, the temperature of the corresponding heating unit TH 21 has to be set to 100 °C.

VIAFLO96 Program

The VIAFLO 96 is a pipetting system, which enables the transfer of 96 samples in a single step. The following table shows how to program the instrument for the extraction procedure A.2: VIAFLO96 (2.4.2)

Step| Action| PositionX| PositionZ| Line1| Line2| Line3| Volume| Speed| Cycles
---|---|---|---|---|---|---|---|---|---
1| Move (X,Z)| 79,5| 192,5|  |  |  |  |  |
2| Prompt|  |  | POSITION| A RACK| B TIP BOX|  |  |
3| Tip Change|  |  |  |  |  |  |  |
4| Move (X,Z)| -80| 192,5|  |  |  |  |  |
5| Prompt|  |  | POSITION B| EMPTY| WASTE BOX|  |  |
6| Move (X,Z)| -80| 90|  |  |  |  |  |
7| Aspirate|  |  |  |  |  | 300| 1|
8| Move (X,Z)| -80| 80|  |  |  |  |  |
9| Aspirate|  |  |  |  |  | 350| 1|
10| Move (X,Z)| 79,5| 192,5|  |  |  |  |  |
11| Move (X,Z)| 79,5| 90|  |  |  |  |  |
12| Purge|  |  |  |  |  |  | 5|
13| Prompt|  |  | POSITION A| REMOVE| RACK|  |  |
14| Prompt|  |  | NEXT| EXTERNAL| STEP 8*|  |  |

Note: *Continue with step 8 of the extraction procedure A.2: VIAFLO96

Step| Action| PositionX| PositionZ| Line1| Line2| Line3| Volume| Speed| Cycles
---|---|---|---|---|---|---|---|---|---
15| TipChange|  |  |  |  |  |  |  |
16| Move (X,Z)| -80| 192,5|  |  |  |  |  |
17| Prompt|  |  | POSITION B| EMPTY| WASTE BOX|  |  |
18| Move (X,Z)| 79,5| 192,5|  |  |  |  |  |
19| Prompt|  |  | POSITION A| RESERVOIR| LYSIS BUF|  |  |
20| Move (X,Z)| -80| 72,5|  |  |  |  |  |
21| Mix|  |  |  |  |  | 250| 5| 10
22| Aspirate|  |  |  |  |  | 300| 1|
23| Move (X,Z)| 79,5| 192,5|  |  |  |  |  |
24| Prompt|  |  | POSITION A| REMOVE| RESERVOIR|  |  |
25| Prompt|  |  | POSITION A| ADD| RACK|  |  |
26| Move (X,Z)| -80| 90|  |  |  |  |  |
27| Dispense|  |  |  |  |  | 300| 10|
28| Move (X,Z)| -80| 80|  |  |  |  |  |
29| Mix|  |  |  |  |  | 300| 10| 10
30| Move (X,Z)| 79,5| 192,5|  |  |  |  |  |
31| Move (X,Z)| 79,5| 90|  |  |  |  |  |
32| Prompt|  |  | NEXT| EXTERNAL| STEP 10|  |  |
33| Prompt|  |  | QUIT AND| REMOVE| TIPS|  |  |

Note: RACK = tube rack

Workflows

Procedure A.1: STANDARD describes the DNA extraction from up to 800 µl enrichment culture using 8-strip tubes and multichannel pipettes. This procedures is a more sensitive protocol.
The A.2: VIAFLO96 protocol includes the VIAFLO96 as a pipetting device, which enables the transfer of 96 samples in a single step.
In the B: RAPID protocol, 100 µl sample is added directly to the pre-aliquoted lysis buffer, without an initial centrifugation step, saving time and effort.

EXTRACTION PROCEDURE A.1: STANDARD

This protocol is intended for extracts that will be used in combination with foodproof® kits.

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

2. 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.

3. ADD SAMPLE
   Transfer up to 800 µl sample (enrichment culture supernatant) to the 8-tube strips.

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

5. CENTRIFUGE RACK
   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 of the used centrifuge.

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 up to 650 µl supernatant carefully with a multichannel pipette immediately after centrifugation, discard and inactivate appropriately. 150 µl have to remain in the tube.
   Take care that the tips of the pipette in the reaction tubes are not touching the pellets.

8. PREPARE LYSIS BUFFER
   Transfer required lysis buffer to a sterile reservoir.
   300 µl lysis buffer per sample plus 1 ml lysis buffer as dead volume.
   Note: Shake the bottle with lysis buffer gently in a short time interval 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 pipette 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 (row 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 450 µl water to balance it out.

12. MECHANICAL DISRUPTION
    Place the 2 adapters in the cell disruption unit and run mechanical disruption
    MPS-1: 8 min at 2,100 rpm.
    Mixer Mill 400: 8 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.
    Weight 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 RACK
    Reinstall tube rack bottom.
    Centrifuge 5 min at 5,400 x g (or 10 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 of the used centrifuge.

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 analysis later on, 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 A.2: VIAFLO96

The following protocol describes the DNA extraction from 800 µl enrichment culture using the VIAFLO 96 instrument. This procedure is equivalent to STANDARD (2.4.1.).

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

2. 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.

3. ADD SAMPLE
   Transfer up to 800 µl sample (enrichment culture supernatant) to the 8-tube strips.

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

5. CENTRIFUGE RACK
   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 of the used centrifuge.

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. START VIAFLO96 PROGRAM
   Switch on the VIAFLO96 instrument. Start program “custom/SPTWO8_A2” and follow the instructions. Run protocol step 1 to step 14.
   Note: Step “Tip Change“ means to discard any used tips, to remove the waste box from position B and to add a tip box at postion B.

8. PREPARE LYSIS BUFFER
   Transfer required lysis buffer to a sterile VIAFLO 96 reservoir.
   300 µl lysis buffer per sample plus 5 ml lysis buffer as dead volume.
   Note: Shake the bottle with lysis buffer gently in a short time interval to avoid sedimentation of ingredients.

9. CONTINUE VIAFLO96 PROGRAM
   Run protocol step 15 to step 33.
   Note: Step “Tip Change“ means to discard any used tips, to remove the waste box from position B and to add a tip box at postion B.

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 (row 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 450 µl water to balance it out.

12. MECHANICAL DISRUPTION
    Place the 2 adapters in the cell disruption unit and run mechanical disruption
    MPS-1: 8 min at 2,100 rpm.
    Mixer Mill 400: 8 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 strips 5 min at 100 °C in TH 21 Heating Block for 8-tube strips.
    Weight 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 RACK
    Reinstall tube rack bottom.
    Centrifuge 5 min at 5,400 x g (or 10 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 of the used centrifuge.

SUPERNATANT FOR DETECTION
Use only 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 analysis later on, 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: RAPID

This protocol is intended for rapid high-throughput extraction in combination with foodproof® kits.

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

2. PREPARE LYSIS BUFFER
   Transfer required lysis buffer to a sterile reservoir.
   300 µl lysis buffer per sample plus 1 ml lysis buffer as dead volume.
   Note: Shake the bottle with lysis buffer gently in a short time interval to avoid sedimentation of ingredients.
   

3. 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.
   

4. ADD LYSIS BUFFER
   Transfer 300 µl lysis buffer with a multichannel pipette to each tube.
   Pipet lysis buffer up and down in reservoir before using to avoid sedimentation of ingredients.
   For this protocol, it is possible to pre-fill up to 12 strips (96 reactions) at once, store at room temperature and use until the end of the shelf life. Close the filled tubes thoroughly with the cap strips from step 3 to avoid evaporation of lysis buffer
   

5. ADD SAMPLE
   Transfer 100 µl sample (enrichment culture supernatant) to the 8-tube strips.
   

6. SEAL TUBES
   Seal the tubes tightly with sterile cap strips.
   

7. 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 (row 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 450 µl water to balance.
   

8. MECHANICAL DISRUPTION
   Place the 2 adapters in the cell disruption unit and run mechanical disruption
   MPS-1: 8 min at 2,100 rpm.
   Mixer Mill 400: 8 min at 30 Hz.
   The efficiency of disruption depends on the mechanical cell disruption unit.
   

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

10. 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.
    

11. CENTRIFUGE RACK
    Reinstall tube rack bottom.
    Centrifuge 5 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 of the used centrifuge.
    
    SUPERNATANT FOR DETECTION
    Use 5 µl extract for the respective foodproof® PCR (Lyo)Kit.
    Note: Strictly avoid transferring fractions of the sediment to the PCR reaction because this might cause PCR inhibition.
    For analysis later on, store DNA at -15 to -25 °C.
    After thawing, mix briefly by vortexing and centrifuge at 2,000 × g for 5 min.
    Note: The sample is not purified. Proteins, RNA, and other materials remain in the sample. Long-term storage or archiving of prepared DNA samples is not recommended.

Troubleshooting

broth.

Repeat DNA extraction with a reduced sample volume.

DNA extract contains too many PCR inhibitors.| Dilute DNA extract, e.g., 1:10, 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.
Enrichment culture contains substances that reduce the DNA extraction efficiency.| Perform a subcultivation or dilution, e.g., 1:10 in fresh enrichment broth.
Sample contains substances that reduce the DNA extraction efficiency.| Reduce the sample volume.
Not enough target organisms in enrichment culture.| Prolong the incubation phase.
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.

ADDITIONAL INFORMATION

Quality Control
All products are regularly monitored by our quality control. You can fi nd the certificate of analysis (CofA) 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”: BIOTECON Diagnostics 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) BIOTECON Diagnostics does not warrant its product against any and all defects when: the defect is as a result of material or workmanship not provided by BIOTECON Diagnostics; 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 fi tness 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) BIOTECON Diagnostics 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 BIOTECON Diagnostics;
(5) BIOTECON Diagnostics 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) BIOTECON Diagnostics 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 BIOTECON Diagnostics GmbH.
HygienaTM is a registered trademark of Hygiena.
Other brand or product names are trademarks of their respective holders.

Reference Number
The reference number and original BIOTECON Diagnostics article number: S 400 17 L.

Change Index
Version 3, November 2019:
New document layout and content.
Version 4, February 2022:
Rebranding.

Support

If you have questions or experience any problems with our products, please contact us:

www.hygiena.com/technical-support-request

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.

foodproof®
StarPrep Two 8-Strip Kit
High-Throughput

Order No. KIT 2301 86
Kit for 480 reactions

Store kit at 15 to 25 °C
For testing of food
and environmental samples

Approval:

Manual:
Version 4, February 2022

Hygiena LLC
CA 93012 Camarillo
USA
www.hygiena.com/technical-support-request

Manufactured by
BIOTECON
Diagnostics GmbH
Hermannswerder 17
14473 Potsdam
Germany
bcd@bc-diagnostics.com
www.bc-diagnostics.com

References

• Support | Hygiena

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