SAMPLE PROCESSING AND PCR SETUP ON THE GS1

Transcription

1 SAMPLE PROCESSING AND PCR SETUP ON THE GS1 For use with the EasyScreen Sample Processing Kit (SP003) and EasyScreen Detection Kit(s). PROCESSING STEPS 1. Turn on and set heat block to 95 C. 2. Add Reagent 1 to Reagent Heat to dissolve. Mix by inversion and vortexing. 4. Dispense 250µl of combined Reagents 1 and 2 into the provided 1.5ml screw cap tubes. This step can be performed by the GS1 using the Reagent Aliquot program. Combined Reagent 1 and 2 can be stored for up to 4 weeks (Room Temp). COMPLETED The deck may be set up as follow: Reagent Container in position µl standard pipette tips in FTR pedestals 1.5ml screw tubes in 50 well pedestal 5. Inoculate combined Reagents 1 and 2 with clinical specimens using provided swabs. Cover the swab with faecal material and scrap excess off the side of the tube. The bristle of the flocked swab should be visible. For solid samples, pre-wet swabs with combined Reagent 1 and 2 before sampling! For liquid samples, 50µl of the sample may be added directly to the combined Reagent 1 and Vortex mix samples for 10sec then pulse centrifuge 7. Incubate inoculated samples for15mins on a heat block (95 C).

2 8. During sample incubation, prepare the GS1. a. Ensure that daily/weekly maintenance has been performed and that the waste containers are emptied. b. Select the Sample Processing icon/protocol from the desktop to start the program. c. Click on the Start ( ) icon d. Input the number of samples to be processed, including the negative control. e. Input barcode details. i. A new WordPad document will open. Scan in the details for each sample. ii. Save the file as Barcode.txt f. Add reagents to each reagent container as guided by the program. g. Arrange the tips, pedestals and plates on the deck as per the guides and deck layout diagram on the computer. h. Select OK to start the program. 2ml Deep well Nunc plate on Heater Shaker 1000µl wide pipette tips in FTR pedestals 1000µl standard pipette tips in FTR pedestals Magnetic separator on DWP pedestal Samples in Microtube pedestal (50 x 1.5ml side) Reagent container in position 1, 2, 3, 4 Elution plate (Hard-shell 96 well plate) on PCR96 adapter on DWP pedestal It is recommended that a new set of reagent containers should be used for each run to ensure nuclease-free consumables. When the run is completed, discard the unused reagents in reagent containers so that nucleases are not introduced into left-over reagents.

3 9. Once incubation is complete, vortex mix samples for 10sec. 10. Spin samples at 13,000xg for 1 minute. 11. Load samples into the 50-well pedestals in the designated order. 12. Ensure that the contents of reagent container #1 are mixed well and the magnetic beads are resuspended. 13. Close the GS1 door and click OK on the computer to start the Sample Processing protocol. 14. Once extraction is completed, remove the elution plate and seal if not using immediately. Discard the used reagent containers.! Spin elution plate for 30 seconds before inoculating eluate into PCR! Nucleic Acid is eluted in this order: Please follow local waste disposal guidelines to discard the waste. All liquid waste generated should be considered hazardous in nature.

4 15. Thaw PCR mastermix and PCR components of the desired EasyScreen Kit. Add the Mastermix component to the PCR components and mix well by vortexing, and then pulse centrifuge µl of mastermix and 1.5ul of the eluted nucleic acid will be transferred into a 384-well PCR plates by the GS1 using the PCR 384 protocol/program. a. Select the PCR384 icon from the desktop to start the protocol. b. Click on the Start ( ) icon. c. Select the barcode file containing the samples to be processed. i. Barcode files will be in Barcode_YYMMDD_Time.txt format. Eg. Barcode_140911_545PM.txt ii. Decide if you want to run a subset of the samples. Select N if you do not want to run a subset Select Y if you want to run a subset of the samples d. Check all the reaction mixes to be processed are loaded on the Microtube pedestal. e. If repeat samples are to be run, select the Repeat Samples check-box. i. Select the barcode file corresponding to the plate to be processed. ii. Scan the barcode corresponding to the repeat samples f. To build the deck, select the Deck Guide check-box. g. Arrange the tips, pedestals, reaction mixes, positive control (Cat. No. PC-ENT-001), elution and repeat plates on the deck as per the guides and deck layout diagram on the computer. h. Select Continue to start the protocol. Repeat samples in Elution plate (Hard-shell 96 well plate) on PCR96 adapter on DWP pedestal Reaction mix in Microtube pedestal (40 x 2.0ml side) 300µl pipette tips in FTR pedestals Fresh samples in Elution plate (Hard-shell 96 well plate) on PCR96 adapter on DWP pedestal 50µl pipette tips in FTR pedestal 384-well PCR plate in 384-well adapter, on DWP pedestal The PCR positive control in the last well of the 40-well pedestal. When using the GS1 to inoculate repeat samples into the 384-well plate, place nuclease-free water in the top right position of the 40-well pedestal.

5 17. If the 384-well plate is dispensed manually, transfer a. 16µl of reaction mix to 384 PCR plates as appropriate. b. 1.5µl of eluted samples into the 16 µl of PCR reaction mix. 18. Seal the 384-well PCR plate with optical film and quick-spin. 19. Amplify as per instructions for real-time PCR machine of choice. REQUIRED CONSUMABLES FOR THE GS1: Item Catalogue number 50µl CO-RE filter tips GS-CORE ul CO-RE filter tips GS-CORE µl CO-RE filter tips GS-CORE µl CO-RE wide filter tips GS-WIDE mL reagent containers GS-CONT-50 Waste bags GS-BAG-100 Nunc 96 deep well plates 2mL, sterile Hard Shell Thin Walled 96-well PCR plates (Biorad) 384-well PCR plates (Roche) and optical film Adhesive films NUN HSP9601, HSP9611, HSP9621, HSP9641 Roche part number O