hygiena KIT 2301 80 foodproof Magnetic Preparation Kit Instruction Manual

hygiene KIT 2301 80 foolproof Magnetic Preparation Kit

Product Information

• The foodproof Magnetic Preparation Kit I is designed for the isolation of bacterial DNA from enrichment cultures of various food samples (raw material and processed food). It is optimized for Gram-negative bacteria and the purified DNA is highly suitable for PCR applications. The kit provides semi-automated purification of total genomic bacterial DNA when used with the foodproof RoboPrep 32 or KingFisherTM Flex instrument.
• The kit contains five bottles of ready-to-use solutions and one bottle of elution buffer. The kit components contain irritating compounds that can be harmful when in contact with skin, inhaled, or swallowed. Always store and use the kit away from food for humans and animals. Wear gloves and follow standard safety precautions during handling.

Kit Components

Product Usage Instructions

Before You Begin

Preparation of working solutions is required in addition to the ready-to-use solutions supplied with the kit. Refer to the manual of Reagent P (Order No. KIT 2300 07) for detailed instructions on preparing the working solution for protein-rich food samples.

How to Use This Product

Product Overview

The foolproof Magnetic Preparation Kit I is designed for the isolation of bacterial DNA from enrichment cultures of various food samples (raw material and processed food). The kit is optimized for Gram-negative bacteria and provides semi-automated purification of total genomic bacterial DNA when used with the foodproof RoboPrep 32 or KingFisherTM Flex instrument.

Test Principle

The basic steps involved in using the foodproof Magnetic Preparation Kit I are:

1. Lysis of cells by incubating with foodproof Magnetic Preparation Kit I Lysis Buffer
2. Binding of DNA to magnetic beads
3. Washing of bound DNA to remove proteins and other cellular impurities
4. Recovery of purified DNA using the foodproof Magnetic Preparation Kit I Elution Buffer

Application

The kit is optimized for enrichment cultures of various food samples (raw material and processed food) with a sample material of 0.2 ml. The kit is optimized for Gram-negative bacteria. The quality of the DNA obtained with the kit is highly suitable for applications using any PCR System. When working with protein-rich food samples like egg, ham, beef, pork, chicken, minced meat, salmon, and cheese, the addition of Reagent P (Order No. KIT 2300 07) to the foolproof Magnetic Preparation Kit I Lysis Buffer is necessary. Refer to the manual of Reagent P for detailed instructions on preparing the working solution for protein-rich food samples.

Procedures and Required Materials

Before you Begin

Preparation of working solutions is required in addition to the ready-to-use solutions supplied with the kit. Refer to the manual of Reagent P for detailed instructions.

Required Materials

• foolproof Magnetic Preparation Kit I
• Enrichment cultures of food samples (raw material and processed food)
• Reagent P (Order No. KIT 2300 07) for protein-rich food samples
• foolproof RoboPrep 32 or KingFisherTM Flex instrument for semi-automated purification of total genomic bacterial DNA

Steps to Use the Product

1. Lysis of Cells: Incubate cells with foolproof Magnetic Preparation Kit I Lysis Buffer.
2. Binding of DNA: Bind DNA to magnetic beads.
3. Washing: Wash bound DNA to remove proteins and other cellular impurities using foolproof Magnetic Preparation Kit I Binding Buffer and Wash Buffer I, II, and III in sequence.
4. Elution: Recover purified DNA using a foolproof Magnetic Preparation Kit I Elution Buffer.

Kit Components

• All solutions except foodproof Magnetic Preparation Kit I Lysis Buffer (bottle 1) and foodproof Magnetic Preparation Kit I Binding Buffer (bottle 2) are clear, and should not be used when precipitates have formed. If precipitates have formed, warm the solutions in a 37°C water bath until the precipitates have dissolved.

Chemical Hazard

• The foodproof Magnetic Preparation Kit I Lysis Buffer (bottle 1), foodproof Magnetic Preparation Kit I Binding Buffer (bottle 2) and foodproof Magnetic Preparation Kit I Wash Buffer I (bottle 3) contain irritating compounds that are harmful when brought in contact with skin, inhaled, or swallowed. Always store and use these kit components away from food for humans and animals. Always wear gloves, and follow standard safety precautions during handling.

Number of Preparations

480 isolations

Storage

• Store the kit at 15 °C to 25 °C through the expiration date printed on the label.
• Note: Inappropriate storage at 2 to 8 °C (refrigerator) or -25 to -15 °C (freezer) will adversely affect nucleic acid purification when precipitates form in the solutions.

Kit Contents

·          205 ml

·          For lysis of cells and extraction of DNA

2 green cap| food proof Magnetic Preparation Kit I Binding Buffer| · 120 ml, add 80 ml absolute isopropanol.

·          For binding of DNA to the magnetic beads

3 blue cap| food proof Magnetic Preparation Kit I Wash Buffer I| · 254 ml, add 154 ml absolute isopropanol

·          For removing impurities

4 blue cap| food proof Magnetic Preparation Kit I Wash Buffer II| · 246 ml, add 164 ml absolute isopropanol

·          For removing impurities

5 blue cap| food proof Magnetic Preparation Kit I Wash Buffer III| · 410 ml

·          For removing impurities

6 white cap| food proof Magnetic Preparation Kit I Elution Buffer| · 192 ml

·          For elution of DNA

How to Use This Product

Product Overview

Test Principle

• The foodproof Magnetic Preparation Kit I in combination with the foodproof RoboPrep+ workstation or the foodproof RoboPrep Fusion system provides fully automated purification of total genomic bacterial DNA from enrichment cultures of food samples. The kit provides high-quality DNA, which is suitable for direct use in PCR applications. The foodproof RoboPrep+ Series workstation or the foodproof RoboPrep Fusion system perform all steps of the sample preparation procedure and can also perform the PCR setup procedure.
• Additionally, the foodproof Magnetic Preparation Kit I in combination with the foodproof RoboPrep 32 or the KingFisherTM Flex instrument provides semi-automated purification of total genomic bacterial DNA from enrichment cultures of food samples.
• The cells are lysed during a short incubation with the provided foodproof Magnetic Preparation Kit I Lysis Buffer. After addition of the foodproof Magnetic Preparation Kit I Binding Buffer the DNA selectively binds to the magnetic beads. Bound DNA is purified in three washing steps to remove potential PCR inhibitors. Then a low-salt elution buffer releases the DNA from the magnetic beads. This simple method eliminates the need for organic-solvent extractions and DNA precipitation, thus providing rapid, simultaneous purification of many samples.

Basic Steps

I Lysis Buffer
2| DNA is bound to magnetic beads
3| Washing of bound DNA to remove proteins, and other cellular impurities
4| Purified DNA is recovered using the food proof Magnetic Preparation Kit I Elution Buffer

Application

• The foodproof Magnetic Preparation Kit I is optimized for isolation of bacterial DNA from enrichment cultures of various food samples (raw material and processed food). The kit is optimized for Gram-negative bacteria. The quality of the DNA obtained with the kit is highly suitable for applications using any PCR System.
• Note: For protein-rich food samples like egg, ham, beef, pork, chicken, minced meat, salmon and cheese the addition of the Reagent P (Order No. KIT 2300 07) to the foodproof Magnetic Preparation Kit I Lysis Buffer is necessary. The preparation of the working solution for this application is described in detail in the manual of the Reagent P.

Sample Material

• 0.2 ml enrichment culture of food samples (raw material and processed food).

Quality Control

• 5 x 104 cfu/ml Salmonella enterica subsp. enterica serovar Senftenberg is extracted and purified as described below.
• 5 µl of the eluate is analyzed using the foodproof Salmonella Detection Kit, 5’Nuclease (Order No. KIT 2300 49). As expected, the resulting amplification signal is obtained.
• Additional DNA preparation and subsequent PCR set up of an unspiked broth sample is used as a negative quality control against contaminating DNA.

Procedures and Required Materials

Before you Begin

Preparation of Kit Working Solutions

• In addition to the ready-to-use solutions supplied with the kit, you will need the following working solutions; preparation of working solutions is required

Bottle| Content| Preparation of a working solution| Storage and stability
---|---|---|---
2 green cap| Binding Buffer| Add 80 ml absolute isopropanol to food proof Magnetic Preparation Kit I Binding Buffer.

Note: Check the box on the label of the bottle after isopropanol has been added. Add the date for verifiability.

| Store at 15 to 25 °C. Stable until the expiry date printed on kit label.
3 blue cap| Wash Buffer I| Add 154 ml absolute isopropanol to food proof Magnetic Preparation Kit I Wash Buffer I.

Note: Check the box on the label of the bottle after isopropanol has been added. Add the date for verifiability.

| Store at 15 to 25 °C. Stable until the expiry date printed on kit label.
4 blue cap| Wash Buffer II| Add 164 ml absolute isopropanol to food proof Magnetic Preparation Kit I Wash Buffer II.

Note: Check the box on the label of the bottle after isopropanol has been added. Add the date for verifiability.

| Store at 15 to 25 °C. Stable until the expiry date printed on kit label.

Protocol for the foodproof RoboPrep+ workstation

Additional Equipment and Reagents required

• foodproof RoboPrep+ workstation
• RAININ disposable filter tips, 250 μl
• RAININ disposable tips, 1000 μl
• RAININ disposable filter tips, 1000 μl
• Eppendorf troughs (reagent containers)
• Process deep well plates
• Elution micro plates
• PCR Cooler for 96-well plate
• 5 ml tubes (Mastermix preparation)
• Disposable waste bags
• 12 ml sample tubes
• PCR reagents
• PCR plates
• Isopropanol, absolute
• Reagent P (Order No. KIT 2300 07), recommended for protein-rich food matrices

Placement of Reagents and Equipment

Deck-Layout foodproof RoboPrep+ 150-8 workstation

Deck-Layout foodproof RoboPrep+ 100-8 workstation

Figure 1. Positioning of reagents and equipment on the deck of the foodproof RoboPrep+ workstations.

1. PCR setup rack
2. Eppendorf Troughs, 6 reagent containers
3. Disposable waste bag
4. Rack for 12 ml sample tubes, up to 96 tubes

• E: Elution microplate
• F: RAININ disposable filter tips, 1000 μl
• no F: RAININ disposable tips, 1000 μl
• P: Process deep well plate
• PCR: P CR plate

Placement Procedure

1. Place the PCR plate, the elution micro plate and the process deep well plate at the appropriate starting positions (PCR, E and P).
2. Place the disposable waste bag at the appropriate position (3).
3. Load the metal racks with tips (1000 μl) (F, no F)

Note: For placement of tips in the workstation pay attention to the motion sequence of the workstation’s pipetting arm during the tip pickup process. It starts with the top right tip rack and ends with the bottom left one (see Fig. 2). Within each tip rack, single tips are picked up the opposite way (see Fig. 3).

Load the sample tubes into the rack (4)
Note: The sample tubes will be used starting with the top left tube and ending with the bottom right tube (see Fig. 4).

Load the reagent containers with the following kit components (3, Fig. 5)

1. Lysis Buffer
2. Binding Buffer
3. Wash Buffer I
4. Wash Buffer II
5. Wash Buffer III
6. Elution Buffer

Load the PCR setup rack with the following PCR kit components (1, Fig. 6)

• White: Tubes with Internal Control.
• Red: Tubes with Enzyme Solution
• Yellow: Tubes with Master Mix
• P: Control Template
• N: Negative control, PCR-grade H2O
• M: 5 ml tube for PCR Mix

Caution

• Always wear gloves during the procedure, and follow safety precautions to minimize contact when handling. Follow all universal safety precautions governing work with biohazardous materials (e.g., wear lab coats at all times). Also, properly dispose of all contaminated materials, decontaminate work surfaces, and use a biosafety cabinet whenever aerosols might be generated.
• The following protocol describes the automated DNA isolation and additional PCR setup from 0.2 ml enrichment culture with the foodproof RoboPrep+ workstation.

1. Place 5 – 10 ml of food enrichment culture into 12 ml sample tubes.

2. Switch on the foodproof RoboPrep+ workstation and monitor and allow the system to boot up.

3. The power switch is on the back side of the instrument.

4. Double-click the lirix3 shortcut icon on the desktop.

5. The log in screen will be displayed (Fig. 7).

6. Enter user name and password.

   • Note that a password was set for all users in the access control manager.

7. Select OK.

8. The screen “Application Setup – Methods” will be displayed (Fig. 8).
   Note that only those categories to which the logged on user has access rights will be displayed and active.

9. Select method “foodproof_MPK_I_Process_vxx”

   • The available methods are grouped under headings.
   • To open a group select the heading required (e.g. “BIOTECON_Processes”). To select a method, left-click on the method name.
   • Choose the method “foodproof_MPK_I_Process_v06” for food samples with low protein content. or
   • Choose the method “foodproof_MPK_I_Process_v07” for protein-rich food samples.

10. Select input variables from the “Input Variables” dialog window (Fig. 9).

11. Choose the number of samples: 1 – 96.

12. Choose the start position.

13. For PCR setup, choose whether a positive and a negative control should be included. If yes, PCR mastermix will be prepared for one positive and one negative control each in addition to the number of samples. If not, PCR mastermix will be prepared for the processed number of samples only.

14. Choose the appropriate volume for the Master Mix according to the used PCR Kit:

    • If you are using a foodproof Detection Kit based on 5’Nuclease technology, choose 18.
    • If you are using a foodproof Detection Kit based on Hybridization Probes technology, choose 13.

15. Choose whether you want to conduct DNA extraction.

16. Choose whether you want to conduct a PCR setup. Click OK to continue.

17. In the “Start Run” dialog window (Fig. 10), you can choose to start the run with a new tip rack. If you do so, tips will be taken starting from the first position of the first tip rack defined in the labware layout. If not, tips will be taken starting from the next available tip after the last used tip position. Note that switching from one method to another with a different process layout resets the used tip position back to the first position in the first rack.

18. The instrument is initialized and the run starts.

19. Place the sample tubes into the sample tube racks.

20. Request for all necessary equipment and reagents.
    The following dialog windows will be displayed and the software will now guide you through the remaining steps required to set up the RoboPrep+ workstation for the “foodproof Magnetic Preparation Kit I Process”.

21. Check for the necessary volume of Lysis Buffer (Fig. 11).
    Fill the necessary volume of Lysis Buffer into the first reagent reservoir and confirm with “continue”.

22. Check for the necessary volume of Binding Buffer (Fig. 12).
    Fill the necessary volume of Binding Buffer into the second reagent reservoir and confirm with “continue”.

23. Check for the necessary volume of Wash Buffer I (Fig. 13).
    Fill the necessary volume of Wash Buffer I into the third reagent reservoir and confirm with “continue”.

24. Check for the necessary volume of Wash Buffer II (Fig. 14).
    Fill the necessary volume of Wash Buffer II into the fourth reagent reservoir and confirm with “continue”.

25. Check for the necessary volume of Wash Buffer III (Fig. 15).
    Fill the necessary volume of Wash Buffer III into the fifth reagent reservoir and confirm with “continue

26. Check for the necessary volume of Elution Buffer (Fig. 16).
    Fill the necessary volume of Elution Buffer into the sixth reagent reservoir and confirm with “continue”.

27. Check for the necessary plates (Fig. 17).
    Place the elution plate, the PCR plate and the process plate at the start positions and confirm with “continue”.

28. Check for the necessary number of racks with filter tips (Fig. 18).
    Place the necessary number of racks with filter tips on the deck beginning with the first tip rack defined in the labware layout and confirm with “continue”.

29. Check for the necessary number of tips without filters (Fig. 19).
    Place the necessary number tips without filters into the defined rack and confirm with “continue”.

30. Check for the PCR reagents (Fig. 20).
    Place the necessary PCR reagents into the PCR setup rack and confirm with “continue”.

31. Check for the 5 ml tube for the Master Mix preparation (Fig. 21).
    Place the 5 ml tube into the PCR setup rack and confirm with “continue ”.

32. All next steps are fully automated, and a software message on the screen will indicate when the protocol is finished (Fig. 22).

Additional Information:

• The protocol steps window shows the list of programmed actions and the running action is highlighted.
• Pause and continue a run: To pause or abort a process, click on the pause icon on the screen or press the pause key. The RoboPrep+ workstation completes the current action before pausing. A dialog then prompts the user to abort or continue the method.

Storage of Samples

Protocol for the foodproof® RoboPrep Fusion system

• The foodproof RoboPrep Fusion is the combination of the JANUS® G3 Integrator instrument from PerkinElmer® and the KingFisherTM Flex instrument from Thermo Fisher Scientific. The foodproof RoboPrep Fusion is controlled by the WinPREP® software of the PE JANUS G3 instrument.

Additional Equipment and Reagents required

• 175 μl Conductive Filter Tips
• 900 μl Conductive Filter Tips
• 1 Well Liquid Handling Reservoir
• 4 Well Liquid Handling Reservoir
• Universal Lid for all 96-well Plates
• 10 ml Sample Tubes, sterile
• KingFisherTM 96 tip comb for DWH
• KingFisherTM 96 Deepwell Plate, sterile
• KingFisherTM 96 Plate, 200 μl
• Sealing foil
• Disposable gloves
• ddH2O
• Vortexer
• Absolute isopropanol (96-98 %)
• All necessary plastic consumables are available through BIOTECON Diagnostics.
• Running the protocol on the foodproof® RoboPrep Fusion

Caution

• Always wear gloves during the procedure, and follow safety precautions to minimize contact when handling. Follow all universal safety precautions governing work with biohazardous materials (e.g., wear lab coats at all times). Also, properly dispose of all contaminated materials, decontaminate work surfaces, and use a biosafety cabinet whenever aerosols might be generated.
• JANUS Application Assistant guides you through the entire process of selecting and running the protocol.
• To open JANUS Application Assistant click on this icon located on your Windows desktop:

A. Daily Preventative Maintenance

• Flushing the VarispanTM system of the foodproof RoboPrep Fusion with degassed distilled water helps to keep the system free of air bubbles, crystals, precipitates, and biological growth that can accumulate within the tubing, valves, and syringes. If allowed to accumulate in the liquid path, these items decrease the accuracy and precision of the instrument. To prevent this problem, flush the system at the start of the working day.

To flush the system:

1. Fill the system liquid container with degassed, distilled water.
2. Clicking on the Select button and choosing the protocol “FlushSysLiq” under the Select Protocol section “Cleaning” of the window to start the flush process.
3. Set both the Flush and Wash volumes to 40,000 μl, when prompted.
4. Clicking on the Run button and the Start button to start the process
5. While the protocol is running, the relative status of the protocol is constantly updated to the screen by the Progress panel.

B. Executing the foodproof MPK I protocol

1. Clicking on the Select button and choosing the protocol “foodproof_MPK_I_vxx” under the Select Protocol section “NA Extraction” of the window.
2. Respond to the questions that are associated with the selected protocol. The questions are listed under the Answer Questions section of the window. You must respond to these questions before you move on to the next step. The answers that you provide help to govern the successful execution of the protocol:
3. Proceed to Step 2: Click on the Next Step button or the Gather button:
4. Inspect the checklist (under the section Gather the Following Labware and Reagents) and collect the labware that you will need to run the protocol. Click on each labware item that you collect. All labware that is listed is required. For your convenience, the location of a lab item or reagent may be listed. This should shorten the time it takes to find the item.
5. Proceed to Step 3. Click on the Next Step button or the Place button:
6. Next, you need to populate the instrument deck with the labware items that you collected in Step 4. The collected labware and reagents are listed under the Place the Following Labware and Reagents section of the window. The Deck Position of each item is also listed. Click on each labware item that you place on the deck:
7. When you select an item in the top left section of the window (Place the Following Labware and Reagents), the item’s placement instructions are displayed under the Instructions section of the window:
   • The bottom portion of the window offers a panoramic view of the entire populated deck:
8. Pipet 5 – 10 ml of the food enrichment culture into a 12 ml sample tube and place it into the sample tube rack.
9. Run the protocol and monitor its progress as the protocol executes.
10. Reset tip boxes or start the tips remaining from a previous run
11. Additionally, the list of labware can be verified
12. Start PCR Setup for LyoKits after decapping the PCR tubes
13. The following window indicates that the protocol is finished
14. Cleanup the instrument after the successful execution of the protocol.

Storage of Samples

The following protocol describes the automated DNA isolation and additional PCR setup from 0.2 ml enrichment culture with the foodproof RoboPrep Fusion:

1. Prefilling of the KingFisherTM Flex plates by the PerkinElmer® JANUS G3 instrument:
   • Elution Plate: 300 μl Elution Buffer added
   • Washing Plate I: 750 μl Wash Buffer I added
   • Washing Plate II : 750 μl Wash Buffer II added
   • Washing Plate III : 750 μl Wash Buffer III added
   • Lysis Plate: 320 μl Lysis buffer (+ 25 μl Reagent P, optional)
2. Transfer of 200 μl sample into the Lysis Plate.
3. Transport of the KingFisherTM Flex plates to the KingFisherTM Flex instrument
4. Execution of the foodproof MPK I program on the KingFisherTM Flex instrument
5. After an elevated lysis step of 10 min a pause step occurs. The Lysis Plate is automatically transported to the PerkinElmer® JANUS G3 instrument.
6. Lysis Plate: 315 μl Binding Buffer added
7. Transport of the Lysis plate to and continuation of the program on the KingFisherTM Flex instrument
8. After finishing the extraction protocol, the Elution Plate containing the extracted DNA and the other plates are transported back to the PerkinElmer® JANUS instrument.
9. Execution of the PCR setup in the PE JANUS instrument
   The program of the KingFisherTM Flex system consists of the following steps:

• Lysis of cells: Cell lysis for 10 min by continuously mixing.
• Binding of the DNA: Automatically sample mixing for 5 min. Magnetic beads separation. Transfer of the magnetic particles to Washing Plate I.
• First Washing: Automatically sample mixing for 1 min. Magnetic beads separation. Transfer of the magnetic particles to Washing Plate II.
• Second Washing: Automatically sample mixing for 1 min. Magnetic beads separation. Transfer of the magnetic particles to Washing Plate III.
• Third Washing: Automatically sample mixing for 20 s. Magnetic beads separation. Transfer of the magnetic beads to the Elution Plate.
• Elution of the DNA: Incubation of magnetic particles in the Elution Buffer for 10 minutes at 90 °C by continuously mixing. Magnetic beads separation. The magnetic beads will automatically be removed and transferred in Washing Plate III (disposal).

Self-programming of the KingFisherTM Flex instrument

Protocol information

• Protocol name: foodproof_MPK_I_v02_Fusion
• Kit name: foodproof MPK I
• Description: KingFisherTM Flex protocol for isolation of genomic DNA from Gram-negative bacteria from enrichment cultures from raw material and food products.

Plate layouts

Dispensed reagents

File Steps

Protocol for the semi-automated DNA extraction with the KingFisherTM Flex

• Additional Equipment and Reagents required
• KingFisherTM Flex instrument
• Pipette and pipette tips
• Disposable gloves
• ddH2O
• Vortexer
• Absolute isopropanol (96-98 %)
• Microtiter 96 DW plate, 2.0 ml
• KingFisherTM 96 KF plate, 200 μl
• Tip Comb 96 DWH
• Adhesive Seal
• All necessary plastic consumables are available through BIOTECON Diagnostics.
• All necessary plastic consumables are available through BIOTECON Diagnostics.

Protocol

Caution

• Always wear gloves during the procedure, and follow safety precautions to minimize contact when handling. Follow generally applicable safety precautions regulating the work with biohazard materials. Properly dispose of all contaminated materials, decontaminate work surfaces, and use a biosafety cabinet whenever aerosols might be generated.
• The following protocol describes the semi-automated DNA isolation from 200 μl sample material with the KingFisherTM Flex instrument:

1. Switch on the KingFisherTM Flex instrument.
   Note: Please read the user manual carefully before starting the purification process with the KingFisherTM Flex instrument! Resuspend the Lysis Buffer and the magnetic beads in the Binding Buffer thoroughly directly before use!

2. Tip Plate: Place the Tip Comb 96 DWH on a Tip Plate (Use one provided Elution Plate (200 μl) as Tip Plate.).

3. Prefill the Lysis Plate, the Washing Plates and the Elution Plate as described below:

4. Lysis Plate: Add 320 μl Lysis Buffer and 25 μl Reagent P (if necessary)

5. Washing Plate I: Add 750 μl Wash Buffer I

6. Washing Plate II: Add 750 μl Wash Buffer II

7. Washing Plate III: Add 750 μl Wash Buffer II

8. Elution Plate: Add 300 μl Elution Buffer

9. Transfer 200 μl of the sample into the Lysis Plate.

10. Choose assay file ”foodproof_MPK_I_vxx” on instrument and press “START”.

11. Follow instructions on the instruments display and load the prefilled buffer plates in the right position. Confirm with “START” after each loading step, the instrument then will provide the next free loading position automatically.

12. When all plates are loaded, press “START” again to initialize the program. The program starts with the lysis of the sample.

13. After an elevated lysis step of 10 min a pause step occurs. The Lysis Plate is automatically moved to the loading position of the instrument. Take out the plate and add

• 315 μl Binding Buffer. Reinsert the plate into the loading position (pay attention to the correct plate orientation, a disregard will result in a failed run) and press the “START” button to continue with the run. From this point on, the instrument will continue with the purification process without any further user interaction.

The following purification steps will run automatically on the KingFisherTM Flex System:

• Lysis of cells: Cell lysis for 10 min by continuously mixing.
• Binding of the DNA: Automatically sample mixing for 5 min. Magnetic beads separation. Transfer of the magnetic particles to Washing Plate I.
• First Washing: Automatically sample mixing for 1 min. Magnetic beads separation. Transfer of the magnetic particles to Washing Plate II.
• Second Washing: Automatically sample mixing for 1 min. Magnetic beads separation. Transfer of the magnetic particles to Washing Plate III.
• Third Washing: Automatically sample mixing for 20 s. Magnetic beads separation. Transfer of the magnetic beads to the Elution Plate.
• Elution of the DNA: Incubation of magnetic particles in the Elution Buffer for 10 minutes at 90 °C by continuously mixing. Magnetic beads separation. The magnetic beads will automatically be removed and transferred in Washing Plate III (disposal).
• Note: After finishing the extraction protocol, the Elution Plate contains the extracted DNA.
• If the extracted DNA contains carryover of magnetic particles, transfer the DNA into a 1.5 ml reaction tube and centrifuge at maximum speed for 1 minute. Transfer the clear supernatant (contains DNA) into a new tube.

Storage of Samples

Self-programming of the KingFisherTM Flex instrument

Protocol information

• Protocol name: foodproof_MPK_I_v01
• Kit name: foodproof MPK I
• Description: KingFisherTM Flex protocol for isolation of genomic DNA from Gram-negative bacteria from enrichment cultures from raw material and food products.

Plate layouts

Dispensed reagents

File Steps

Protocol for the DNA extraction with the foodproof RoboPrep 32 instrument

• Additional Equipment and Reagents required
• foodproof RoboPrep 32 instrument
• Pipette and pipette tips
• Disposable gloves
• ddH2O
• Vortexer
• Absolute isopropanol (96-98 %)
• 96 Deep Well Plate
• Tip Comb

Protocol

Caution

• Always wear gloves during the procedure, and follow safety precautions to minimize contact when handling. Follow generally applicable safety precautions regulating the work with biohazard materials. Properly dispose of all contaminated materials, decontaminate work surfaces, and use a biosafety cabinet whenever aerosols might be generated.

The following protocol describes the semi-automated DNA isolation from 200 μl sample material with the foodproof RoboPrep 32 instrument:

1. Switch on the foodproof RoboPrep 32 instrument.

2. Open the door and push the lever forward, the temperature-controlling module will sink in.

3. Set up the Tip Combs on the mixing sleeve track.
   Note: Please read the user manual carefully before starting the purification process with the foodproof RoboPrep 32 instrument! Resuspend the Lysis Buffer and the magnetic beads in the Binding Buffer thoroughly directly before use!

4. 96 Deep Well Plate setup and reagent volumes:

1 or 7

| Lysis Buffer| 320
Reagent P (if necessary)| 25
2 or 8| Wash Buffer I| 750
3 or 9| Wash Buffer II| 750
4 or 10| Wash Buffer III| 750
6 or 12| Elution Buffer| 300

1. Transfer 200 μl of the sample into the wells of the 1st or 7th column.
2. Place the 96 Deep Well Plate into the instrument.
3. Pull the lever towards you, the temperature-controlling module will rise up, and tightly connect to the 96 Deep Well Plates.
4. Select protocol for MPK I
5. After finishing the extraction protocol, the column 6 or 12 contains the extracted DNA. Transfer the DNA into a 1.5 ml reaction tube and centrifuge at maximum speed for 1 minute. Transfer the clear supernatant (contains DNA) into a new tube.
6. Then it is recommended to clean the instrument by 70 % ethanol and disinfecting it via UV light program in the apparatus.

The following purification steps will run automatically on the instrument:

• Lysis of cells: Cell lysis for 15 min by continuously mixing.
• Binding of the DNA: Automatically sample mixing for 15 min. Magnetic beads separation. Transfer of the magnetic particles to Wash Buffer I.
• First Washing: Automatically sample mixing for 1 min. Magnetic beads separation. Transfer of the magnetic particles to Wash Buffer II.
• Second Washing: Automatically sample mixing for 1 min. Magnetic beads separation. Transfer of the magnetic particles to Wash Buffer III.
• Third Washing: Automatically sample mixing for 30 s. Magnetic beads separation. Transfer of the magnetic beads to the Elution Buffer.
• Elution of the DNA: Incubation of magnetic particles in the Elution Buffer for 15 minutes by continuously mixing. Magnetic beads separation. The magnetic beads will automatically be removed and transferred in Wash Buffer III (disposal).

Storage of Samples

Self-programming of the foodproof RoboPrep 32 instrument

Edit and run the experiment program as follow:

RUN| Well No. (0-6)| Name| Standby (0-30Min)| Mix

(1-30Min)

| Volume

(100-1000µl)

| Mix Speed (1-3)| Mag

(0-120Sec)

| Temp. (40-80°C)| Pause
---|---|---|---|---|---|---|---|---|---
☒| 1| Lysis| 0| 15| 520| 3| 0| 80| ☒
☒| 1| Binding| 0| 15| 835| 3| 30| 0| ☐
☒| 1| Binding| 0| 0| 835| 0| 30| 0| ☐
☒| 2| Wash-1| 0| 3| 750| 3| 30| 0| ☐
☒| 2| Wash-1| 0| 0| 750| 0| 30| 0| ☐
☒| 3| Wash-2| 0| 3| 750| 3| 30| 0| ☐
☒| 3| Wash-2| 0| 0| 750| 0| 30| 0| ☐
☒| 4| Wash-3| 0| 1| 750| 3| 30| 0| ☐
☒| 6| Elution| 0| 15| 300| 3| 60| 80| ☐
☒| 4| Waste| 0| 1| 750| 3| 0| 0| ☐

Typical Results

Purity
Purified DNA is free of other cellular components and DNA polymerase inhibitors.

Troubleshooting

kit at 15 to 25 °C at all times upon arrival.
Buffer or other reagents were exposed to conditions that reduced their effectiveness.| ·          Store all buffers at 15 to 25 °C.

·          Close all reagent bottles tightly after each use to preserve pH and stability, and to prevent contamination.

·          After any lyophilized reagent is reconstituted, aliquot it, then store the aliquots at

-25 to -15 °C.

Isopropanol not added to food proof Magnetic Preparation Kit I Binding Buffer, Wash Buffer I or Wash Buffer II.| ·          Add absolute isopropanol to the food proof Magnetic Preparation Kit I Binding Buffer, Wash Buffer I and Wash Buffer II before using.

·          After adding isopropanol, mix the food proof Magnetic Preparation Kit I Binding Buffer, Wash Buffer I and Wash Buffer II well, and store at 15 to 25 °C.

·          Always mark food proof Magnetic Preparation Kit I Binding Buffer, Wash Buffer I and Wash Buffer II bottle to indicate the addition of isopropanol.

Reagents and samples not completely mixed.| Always mix the sample tube well after addition of each reagent.
Not enough target organisms in enrichment culture.| Prolong the incubation phase.

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 if the product is contaminated by improper storage or handling;
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. 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.

Supplementary Information

Ordering Information
HygienaTM is offering a broad range of reagents and services. For a complete overview and for more information, please visit our website at www.hygiena.com.
Trademarks

• foodproof® is a trademark of BIOTECON Diagnostics GmbH.
• HygienaTM is a registered trademark of Hygiena.
• JANUS®, WinPREP®, and VarispanTM are trademarks of PerkinElmer, Inc.
• KingFisherTM is a trademark of Thermo Fisher Scientific
• Other brand or product names are trademarks of their respective holders.

Contact and Support

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

Reference Number

• The reference number and original BIOTECON Diagnostics article number: S 400 11 L.

Change Index

Version 6
New document layout and content. New order number.

• 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

Documents / Resources

| hygiena KIT 2301 80 foodproof Magnetic Preparation Kit [pdf] Instruction Manual
KIT 2301 80, KIT 2301 80 foodproof Magnetic Preparation Kit, foodproof Magnetic Preparation Kit, Magnetic Preparation Kit
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References

• Home | Hygiena
• Support | Hygiena

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