STANDARD OPERATING PROCEDURE

Transcription

1 Document No: LAB/023/2 Page 1 of 7 Uncontrolled copy if not printed on yellow paper. Title: STANDARD OPERATING PROCEDURE Use of the Magtration System 8Lx Document No: LAB/023 Version No: 2 CopyNo/Holder: 09/Jay Brown Issue Date: 07/09/07 Review Date: 07/09/09 Issued by: Jay Brown 1 INTRODUCTION 1.1 The Magtration System 8Lx is a desktop automated nucleic acid purification system that uses magnetic beads to extract DNA from up to 7 ml of whole blood. 1.2 Up to 8 blood samples can be processed simultaneously in 1 hour. 1.3 The Extracted DNA is eluted into 1ml of 10 mm Tris-HCl, ph SCOPE 2.1 This SOP describes the procedures for using the Magtration System 8Lx to extract DNA from up to 7ml of whole blood, which has been treated with an anticoagulant such as EDTA. 3 RESPONSIBILITIES 3.1 It is the responsibility of the laboratory personnel carrying out the procedure to ensure that all steps are completed both competently and safely. 4 RELATED DOCUMENTS 4.1 Disposal of waste is described in the SOP LAB/ Handling of Reagents is described in SOP REA/ Any SOP deviations are recorded as described in the SOP QAU/ Allocating samples to locations is described in SOP LAB/ Maintenance is recorded on Form LAB/ A list of Designated Trainers is recorded on Approved List PER/005C 5 HEALTH AND SAFETY 5.1 All handling of blood samples where there is potential of exposure to blood (e.g. transfer from client tubes) must be performed in a Class II laminar flow cabinet equipped with a drip tray.

2 Document No: LAB/023/2 Page 2 of A lab coat and gloves must be worn. 5.3 All staff working with blood are advised to have Hepatitis B vaccinations. 5.4 Care should be taken when using the Magtration System 8Lx as some components move out and there can be a risk of collision. Ensure that other laboratory users are aware of this before starting the program. 5.5 The reagent kit contains protein denaturants. Wear gloves, and take appropriate safety precautions when using this kit, to avoid direct skin contact. 6 EQUIPMENT/MATERIALS/REAGENTS 6.1 Equipment Equipment Manufacturer CIGMR ID# Magtration System 8LX PSS 0495 Variable volume pipette (P1000) Gilson N/A MagnaRack Invitrogen N/A 6.2 Materials Material Supplier Cat# Blood sample(s) for extraction N/A N/A Syringe PSS F ml Teklab blood tube Teklab Z ml Sarstedt tube Sarstedt Sarstedt elution tube Sarstedt ml cryovial Greiner Reagents Reagent Supplier Cat# Magtration reagent pack PSS E % Gigasept S&M SYSTEM BOOKING 7.1 To book the Magtration System 8Lx, users will send an to eightlxbooking@listserve.manchester.ac.uk stating the time and date they want to book. The number of samples to be processed must be included in the , along with the CIGMR Project Code to be charged. 7.2 Confirmation of the booking will be received via . Reagent packs will be provided on the day of the booking. The number of reagent packs available will be equal to the number of samples specified in the booking .

3 Document No: LAB/023/2 Page 3 of LX users should also book a Class II cabinet in order to perform the blood transfer at an appropriate time (Section 8.1). Booking forms for Cabinet 1 & 2 can be found on the network drive N:\Projects\CIGMR LAB BOOKINGS\Class II Cabinets. 8 DNA EXTRACTION PROCEDURE 8.1 Defrost 1-8 whole blood samples, and in a Class II laminar flow cabinet. Transfer a maximum of 7ml of each blood sample to a labelled 10ml Teklab blood tube. All blood contaminated plastic must be fully submerged in 12% Gigasept overnight, prior to disposal in a Biohazard incineration container 8.2 Before using the Magtration System 8Lx, open the front cover by sliding it upwards, and clean the internal sample stage and tube racks with 70% ethanol as described in section Close the front cover. 8.4 Turn on the Magtration System 8Lx by using the main power switch located on the left side of the machine. 8.5 Open the lower front door located on the front side of the Magtration System 8Lx and turn on the power of the PC by pressing the blue switch located in the centre of the unit. The PC will boot Windows and automatically open the 8Lx system s software. 8.6 A welcome screen will be displayed, showing the following information: User User ID User Level Last Login Last Protocol Last status (displays the error code produced last session, if no errors were produced it displays a 0) Total operating time Last batch ID Total UV time Total Tip On time Total piercing time 8.7 Using the stylus pen on the touch screen select Ok to enter the program. 8.8 Using the stylus pen select Ext.Menu, to display the Ext.Menu screen. 8.9 Select the System Initialization option. The 8Lx sets all the units to their starting home positions Select the Cleaning pins option. The Piercing stage will move to the front of the machine. Open the Front cover and check that each of the piercing pins are in their holes located behind the black magnets. Close the front cover and select Ok. The piercing stage will move back into position.

4 Document No: LAB/023/2 Page 4 of Press the Main Menu button, and then select the Protocol button located in the top left corner of the screen From the new window select Whole Blood for 7ml ver 1.9. Press Ok Select Set Tips. The Z-unit that holds the tips moves forward and the syringe plunger head holder opens up Check on the touch screen that the door is registered as unlocked by looking for a red square in the middle of the screen that says Unlock Open the front of the Magtration System 8Lx. Holding the Syringe tip by the large main body section with the tip pointing down. Insert the lower portion of the syringe tip into the lower hook, then push the upper portion of the syringe tip into the upper hook. Once all tips have been inserted, one for each sample being processed, close the front cover and press Ok. The Z-unit moves to its home position Open the front cover. Use the handle located on the underside of the sample stage to pull the stage forward Insert one 30 ml Sarstedt tube per sample into the heater block (second hole from the back) Remove the sample rack to allow you to insert the pre-packed reagent cartridges. The arrows on the packs must point towards the back of the instrument. Use one cartridge per sample being extracted Remove and retain the lid from a Sarstedt elution tube and insert one tube per sample into the first hole (at the front) on the sample rack Replace the sample racks making sure that R is on the right, and L is on the left. Care must be taken to ensure the sample racks are correctly positioned (in particular, racks must lie completely flat). Remove the lids from the 10 ml Teklab blood tubes and place them into the third hole (towards the back) on the sample rack. If using barcodes line them up with the notches on the sample rack Push the sample stage back into the Magtration System 8Lx, and close the front cover On the touch screen select Scan Info. The barcodes on the samples will now be read. If the barcodes can not be read, or you are not using barcodes, enter sample information manually be selecting the cell you wish to edit on the touch screen, and then select Edit Info Using the On-screen Keyboard input the sample information and press Enter. Repeat 9.22 and 9.23 for other samples if necessary To begin the extraction process press Start. Then select Start Batch. Tips must be completely aligned with the pins. If not, press Pause Batch, open the cover, and gently align the bottom of the tips over the pins. Close the lid and press Start Batch. This can be repeated until the tip covers are ejected. If the tips are not aligned, the tip covers will not eject correctly and the program will abort (characterised by a loud grinding noise). The robot must immediately be switched off and a Designated Trainer notified should this occur.

5 Document No: LAB/023/2 Page 5 of The extraction process will start and is completed in 1 hour Once the method has finished successfully the screen will display Writing data of result date into USB key Memory. Select Ok. This will save the information of the current batch to the hard drive. For retrieving data see 9.39 to On the touch screen select the Unlock option Open the front cover and move the sample stage forwards, as described in Remove the elution tube and screw on the lid. Transfer the tube to the Invitrogen MagnaRack. Leave on the rack for a minimum of 30 minutes to allow any residual magnetic beads to bind to the magnet With the tube still positioned in the MagnaRack, remove the lid and carefully transfer the DNA solution to an appropriate clean tube (e;g 1.8ml cryovial for downstream processing on the Hamilton robot). Tubes must be labelled prior to DNA transfer Remove the 10 ml blood tubes, replace their lids and dispose of them in a Biohazard incineration container Remove the pre-packed reagent cartons and transfer them to a Class II cabinet. Pour their contents into a waste container containing 12% Gigasept and leave overnight before discarding down the sink. Dispose of the empty reagent packs in a Biohazard incineration container. The same procedure is followed for any waste produced in Section Close the sample stage and front cover. On the touch screen select Remove Tips. The Z- unit will move forward Open the front cover Hold the syringe tip below the lower hook. Press the lower hook down and gently pull the syringe tip forward. Then pull the upper section of the syringe tip out of the upper hook. Dispose of the syringe in the Biohazard incineration container Open the cover and remove the 30 ml Sarstedt tubes from the heater block and dispose of them in a Biohazard incineration container. Use caution as the heater blocks will be hot Close the front cover and select Ok. The extraction process is now complete Check for any spillages and clean according to 9.3 if necessary In the event of error codes being produced contact a Designated Trainer for advice To retrieve data from the Magtration System 8Lx, select Ext. Menu. Then select Result File Files are recorded by date (written backwards) and batch e.g _0000.dat would be batch 1 on 1st January Highlight the result file you wish to look at and press Select. The result information for that batch will now be displayed in the table at the bottom of the screen.

6 Document No: LAB/023/2 Page 6 of Before shutting down the system at the end of the day repeat step 10.3 and carry out the sterilization procedure described in SYSTEM MAINTENANCE 9.1 Sterilisation procedure. At the end of each day s use the Magtration System 8Lx needs to be sterilized using the built-in germicidal UV lamp From the main screen select Sterilize The sterilize screen will appear. Use the arrows located in the bottom right corner to adjust the sterilization time to 2 hours Press Start The UV lamp will run for 2 hours, the Magtration System 8Lx will then automatically shut down. The robot should be turned off using the switch on the left-hand side after use To disable the lamp before the sterilization process is complete press Cancel. 9.2 Piercing pin cleaning. Once a month the Piercing pins need to be cleaned and the process must be signed off on the Magtration System 8Lx maintenance log (Form LAB/007) which is located on the side of the Magtration System 8Lx Whilst the front cover is closed go to the main screen and select Ext.Menu Select Cleaning Pins. The pin stage will move forward Open the front cover and remove one piercing pin Use a soft cloth dampened with 70% ethanol to clean the pin Return the pin to its original location Repeat steps to to clean all 8 pins Close the front cover and press Ok. The piercing stage will move back to its home position. 9.3 Stage and magnet cleaning. Once a month the stage and magnets need to be cleaned and the process must be signed off on the Magtration System 8Lx maintenance log (Form LAB/007) which is located on the side of the Magtration System 8Lx Before cleaning the stage and magnets, wait at least 30 minutes after extraction operations have been performed. This will allow the incubation areas to cool Do not allow magnetic cards or metal objects get close to the magnet areas Open the front cover.

7 Document No: LAB/023/2 Page 7 of Dampen a cloth with 70% ethanol and wipe the incubator stage, including inside the incubator holes Wipe the black magnet areas Close the front cover Record that the procedure has been carried out on the Magtration System 8Lx maintenance log (Form LAB/007) which is located on the side of the Magtration System 8Lx. 10 DOCUMENT HISTORY This is the second version of this SOP. The following changes were made: Overall re-write Re-formatting Pin alignment check in 8.22