Set-up, programming and analysis MagNA Pure 96 (Roche)

Transcription

1 Set-up, programming and analysis MagNA Pure 96 (Roche)

2 A. Start-up Before preparing the run, start the instrument and the control unit v

3 1. Starting the MagNA Pure 96 Software To start and log on to the MagNA Pure 96 Software Switch on the instrument by pressing the main switch located on the bottom right side of the instrument. After successful initialization procedure, the LED on the right of the front panel glows green, indicating that the instrument is ready for run preparation (4-5 min). Switch on the monitor and control unit to start the Windows XP operating system (2min). Double-click the MagNA Pure 96 Software icon on the desktop. The initializing status pops up on the screen, followed by display of the main window with the Overview tab. Click Log on in the global action bar: By default : User ID: admin Password: MagNAPure96

4 2. Preparing the MagNA Pure System On daily basis, perform the following for startup of the instrument : Select the Instrument tab. Select Maintenance & Service sub-tab. In the tree-view select Daily Maintenance and below select the check box in front of Prime. Click Start. Click Start Select the check box in front of Confirm Clean Instrument. Click Start. Select the check box in front of Confirm Daily Maintenance. Click Start. When check points are finished, the entry will be marked.

5 B. Preparation of samples Setting up a run for purification of genomic DNA from 96 samples of 200µl using the MagNA Pure 96 DNA and Viral RNA Small Volume Kit v

6 1. Preparing the Internal Control (IC) Adding the internal control to each of the samples and to the negative control is a very important step to see if the nucleic acid isolation has been successful and to check for possible PCR inhibition. FTD recommends to add a minimum of 2μl of FTD IC when the starting volume for the extraction is 200μl and the elution volume is 55μl. However, the MagNA Pure 96 Instrument cannot dispatch such a small amount. The instrument is programmed to add automatically 20μl of IC to each sample. Solution: dilute the IC 1/10 in ddh 2 O according to this table: For example, for 96 samples, mix: 310μl of IC 2790μl of ddh 2 O Remove the cover from the MagNA Pure 96 Internal Control Tube and pipette the 3100μl of the FTD IC diluted 1/10 into it. Close the tube. Number of Samples IC Volume required by MP96 FTD IC ddh 2 O The instrument calculates the total amount of IC needed according to the number of samples on the 96 well plate.

7 The Internal Control Configuration dialog box opens. In the Name field, enter a name for the new IC (i.e. FTD IC or IC3) Click in the Protocols list and select the DNA universal pathogens protocol to which the IC is applied. Click OK to save your settings. In the Settings dialog box, select your new IC and click the barcode icon on the right. The Edit Internal Control Barcode dialog box opens so that you can insert your IC identification number. In the MagNA Pure 96 Software, click Settings in the global action bar. Settings dialog box opens In the tree view, select Internal Controls and click the icon on the right.

8 2. Preparing the Samples Ensure that samples are thawed to heart and well homogenized Pipette 200μl of sample into the individual wells of a processing cartridge (source plate) Pipette the samples into the bottom of the wells using aerosol-preventive tips. Ensure that the source plate is free of droplets on the walls of the wells. Ensure that the samples have reached room temperature before the run.

9 C. Defining parameters in the Software Provide the software with information on protocol and sample parameters v

10 1. Defining order In the Overview tab click [Enter order] 1. Fill in the Orders tab information: Chose Purification 2.Select your extraction kit (DNA/Viral NA in our case) 3.Select DNA Universal Pathogens protocol. 4.Mention the input and output volumes for your extraction (200μl > 50μl) 5.Select FTD IC from the Internal Control list. 6.There is no custom source plate in our case 7.Mention where eluate should be dispatched (MP96 Output plate in our case)

11 Samples Specify the sample data in the table with the names and positions of all specimen placed in the source plate 1. Enter sample names, scan barcodes or load from template 2. Save Order and enter file name 3. When order file is saved in the database, click here

12 3. Stage sub-tab Requirements for the order are displayed

13 D. Loading the Stage v

14 1. Loading the Bottle Rack The bottle rack holds the bottled isolation reagents needed for the order defined on the Orders sub-tab and the internal control tube. The Stage sub-tab displays the relevant reagent bottles to be placed in the bottle rack Open the load flap of the MagNA Pure 96 Instrument, pull the bottle rack all the way out of the stage. Pull back the clip of the rearmost bottle holder (position 1) and insert the first MGP bottle. Press the bottle firmly into the holder until the clip slips over the bottle shoulder. Insert a second MGP bottle in position 2 in the same way. Ensure that the barcode faces left Ensure that the clips fix the bottle rims completely

15 1. Loading the Bottle Rack Remove the cap from the prepared internal control tube and insert it in position 9 of the bottle rack. Ensure that the clips fix the bottle rims completely Insert the bottle rack back into the stage and press the bottle rack firmly until its outline is displayed in blue on the Stage sub-tab.

16 1. Loading the Reagent Rack The reagent racks hold the processing cartridges and the reagent trays needed for the defined order. Pull the left reagent rack all the way out of the stage. Place a processing cartridge in position 1 of the left reagent rack, barcode facing left. Insert the left reagent rack back into the stage and press it in until its outline is displayed in blue on the Stage sub-tab. Pull the right reagent rack all the way out of the stage. Place a processing cartridge in position 1 of the right reagent rack, barcode facing left. Remove the blue lid from reagent trays and place reagent tray 1 in position 2 and reagent tray 2 in position 3 of the reagent rack. Insert the right reagent rack back into the stage. Press the reagent rack into the stage until its outline is displayed in blue on the Stage sub-tab.

17 1. Loading the Tip Rack The tip rack provides the necessary tips packed in tip trays for a purification run. Pull the tip rack all the way out of the stage. Place three tip trays in the tip rack, making sure that all tips stand parallel and in one plane and barcodes face left. You do not need to remove a partially used tip tray from the tip rack. Insert the tip rack back into the stage. Press the tip rack into the stage until its outline is displayed in blue on the Stage sub-tab.

18 1. Loading the Purification Rack The purification rack contains a processing cartridge with the samples, called source plate, in which the purification takes place and the output plate, also called target plate. Pull the purification rack all the way out of the stage and place an empty MagNA Pure 96 Output Plate in position 4 of the purification rack, barcode facing left. Place the processing cartridge with the prepipetted samples in position 1 of the purification rack, barcode facing left. Insert the purification rack carefully so as not to spill the samples onto the stage and press it until its outline is displayed in blue on the Stage subtab. Source plate with pre-pipetted samples Output plate for the purified DNA/RNA

19 1. Loading the Waste Rack The waste rack is used to collect the liquid waste and used tips. Pull the waste rack all the way out of the stage. Ensure that the waste cover is clean. Place the waste cover in position 1 of the waste rack. Place three empty tip trays in positions 2,3, and 4 of the waste rack. When using empty tip trays with barcodes, ensure that the barcodes are facing the right side of the waste rack. Insert the waste rack back into the stage. Press the waste rack into the stage until its outline is displayed in blue on the Stage sub-tab. Close the load flap.

20 E. Performing a run v

21 1. Resource Check A few seconds after the load flap is closed, the MagNA Pure 96 Instrument locks and starts the resource check. The resource check ensures that: All required accessories for the defined run are present. All required disposables for the defined run are present and valid. There is enough system fluid for a run of up to 96 samples. There is enough capacity in the waste container for a run of up to 96 samples. If during the resource check the MagNA Pure 96 Instrument detects that an accessory or disposable is missing, invalid or in the wrong position, a message is displayed. The graph on the Stage sub-tab is color-coded: After the resource check, the MagNA Pure 96 Instrument unlocks the load flap. If positions on the Stage sub-tab are displayed in yellow or red, rearrange or exchange relevant component.

22 Once the resource check is successfully completed, the Stage sub-tab of the MagNA Pure 96 Software looks as follows: Click Start Click OK

23 2. Monitoring the Run The load check ensures that empty tip trays are present in the waste rack, the waste cover is present in the waste rack, the tip park position is empty. Display with the Overview: The start time, the estimated end time of the run and a progress bar indicating the current state of the run

24 3. End of a Run The duration of a purification run depends on the selected purification protocol not on the number of samples processed. Click OK Click See Results A message box appears, notifying that the run has finished. Click OK. Open the flap. Overview indicates the used disposables.

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