TITLE: Semi-Automated Extraction of Viral RNA using the MagMax Viral Pathogen (MVPII) 96 Kit for SARS-CoV-2 Detection
AUTHORS: Pedro Belda-Ferre, Lisa Marotz, Greg Humphrey, Sydney Morgan, Rob Knight

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to describe a semi-automated process for viral RNA isolation from anterior nares (AN) swabs in molecular transport medium (MTM), Mawi medium, or 5% sodium dodecyl sulfate (SDS), using the MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit.</div><div class = "text-block"><span>This procedure applies specifically to samples received for SARS-CoV-2 testing. The MagMAX™ Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit uses magnetic bead technology to rapidly isolate, wash, purify, and elute viral RNA from viral samples in transport media. This process is developed for semi-automated systems and performs RNA extraction in a 96-well plate format. The RNA extracted will be used for SARS-CoV-2 detection via a TaqPath COVID-19 Multiplex Real-Time RT-PCR assay (see related </span><a href="https://www.protocols.io/edit/semi-automated-and-miniaturized-sars-cov-2-detecti-brhvm366" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocol</span></a><span>).</span></div><div class = "text-block">Viral samples are obtained from anterior nares (AN) swabs and placed into MTM, Mawi, or 5% SDS solutions. Viral RNA from these samples is extracted using an Eppendorf epMotion 5075 automated liquid handling workstation and a KingFisher Flex benchtop automated sample preparation machine.</div></div>

[STEPS]
?. [Sample Extraction Plate (SEP) Preparation]
Transfer proteinase K into KingFisher deep-well 96-well plates using the epMotion 5075:  Turn on the epMotion 5075.Open the epBlue software (version 40.7) on the associated laptop, enter username and password.Within the epBlue program, double click to open the 'Application Runner'.Open the following program:
?. [Sample Accessioning and Plating using Hamilton STAR]
In this step,   from each nasal swab sample is added to the wells of the Sample Extraction Plate  (SEP) prepared in Step 2 above using a Hamilton Microlab STAR liquid handling system.Samples are plated into 96-well sample extraction plates (SEP) in batches of 94, with 2 clean media controls in positions A4 and H3 of the SEP (these wells will become the Negative and Positive Control wells, respectively).
Alternatively, this step can be done by hand, by adding   from each sample to the wells of the SEP (containing   Proteinase K) prepared in Step 2. If conducting this procedure by hand, remember to add   clean media to wells A4 and H3 (the control wells) instead of adding any sample and track the samples in a platemap.
?. [Wash and RNA Elution Plate (REP) Preparation for RNA Extraction]
Prepare wash and elution reservoirs:Prepare the following 100 mL reservoirs in a Biological Safety Cabinet:Label one 100 mL reservoir "Wash Buffer" and fill with  wash buffer . Load into Rack 1, spot 1 of epMotion 5075.Label one 100 mL reservoir "EtOH" and fill with  80% ethanol. Load into Rack 1, spot 2 of epMotion 5075.Label one 10 mL reservoir "H2O" and fill with  nuclease-free water stored at . Load into Rack 1, spot 3 of epMotion 5075.
For each SEP containing 94 samples + 2 controls (full 96-well plate), you will need: one (1) KingFisher 96-well deep well plate containing Wash Buffer; one (1) KingFisher 96-well deep well plate containing 80% EtOH; one (1) KingFisher 96-well deep well plate containing Nuclease-Free Water (this will be the RNA Elution Plate [REP]). These plates can be prepared at the same time as the patient sample plating (section 4) above.
50 mL
100 mL
10 mL
0 Room temperature
?. [Binding Bead Mix Solution Addition to SEP]
Prepare binding bead mix solution:Place a 100 mL epMotion reservoir into the Reservoir Rack in the top-left slot (position 1) and add  of MagMAX Binding Solution  using a serological pipette. Thoroughly vortex the MVPII Binding Beads and add  using a P1000 pipette. Mix the solution by pipetting up and down while moving pipette tip sideways back and forth, until solution is homogenous.
Add binding bead mix only after sample has been added to SEP (section 4 above).
MVPII Binding Beads tend to clump, and must be thoroughly vortexed until no clumps adhere to walls of bottle when rotated. This is to ensure even distribution of beads among the samples.
27 mL
1020 µl
?. [MS2 Phage Control Addition]
Add MS2 Phage to the SEP:Thaw one aliquot of 1:160 diluted MS2 phage control.If necessary, turn on the epMotion 5075 and open the eBlue software on the associated laptop, enter username and password.Load the following epMotion protocol:
The MS2 phage is an internal positive control that serves as an extraction, reverse transcription, and qPCR positive control for each well. If only the MS2 phage amplifies, the sample is negative. If nothing amplifies (not even the MS2 phage control), this means that something went wrong with the extraction or with the RT-qPCR, and the sample is labelled as “invalid” and must be extracted again.This step will add   of MS2 to every well of the SEP except for the positive control well, A4.
If necessary, prepare the 1:160 diluted MS2 phage aliquots:Retrieve MS2 phage from the freezer and the nuclease-free water from benchtop storage.In a biosafety cabinet, make a   solution of 1:160 diluted MS2 phage in a sterile 50 mL conical tube by combining   nuclease-free water and   MS2 phage control (Thermo, PN: 100092698).Transfer   of the diluted MS2 phage solution from the 50 mL conical tube into sterile 2.0 mL microcentrifuge tubes labelled with "MS2" and the aliquot preparation date. This solution will make 33 aliquots.Store MS2 phage aliquots in the freezer at  .
?. [Viral RNA Extraction using KingFisher Flex]
Open INSPECT application if set up for your lab.If a data tracking application for SARS-CoV-2 tests is desired, this application can be set up for any lab following the attached protocol: dx.doi.org/10.17504/protocols.io.bis8kehwScan in plates and reagent lot numbers to INSPECT App:In the INSPECT app, click the “qRT-PCR” button on the left side:  In the following page, click “RNA elution”:  In the following page, use the pull-down menu to select the appropriate lot numbers used for the MS2 Phage, Wash buffer, Binding Solution, EtOH (used in the second wash plate), and nuclease-free water (used in the elution plate).  Place your cursor in the “Sample Extraction Plate Barcode” section and scan in the plate barcode affixed to the SEP.Place your cursor in the “RNA Elution Plate Barcode” section and scan in the plate barcode affixed to the REP.Click “Assign Plates”.In the following page, click “RNA elution”:  In the following page, use the pull-down menu to select the appropriate lot numbers used for the MS2 Phage, Wash buffer, Binding Solution, EtOH (used in the second wash plate), and nuclease-free water (used in the elution plate).  Place your cursor in the “Sample Extraction Plate Barcode” section and scan in the plate barcode affixed to the SEP.Place your cursor in the “RNA Elution Plate Barcode” section and scan in the plate barcode affixed to the REP.Click “Assign Plates”.In the following page, use the pull-down menu to select the appropriate lot numbers used for the MS2 Phage, Wash buffer, Binding Solution, EtOH (used in the second wash plate), and nuclease-free water (used in the elution plate).  Place your cursor in the “Sample Extraction Plate Barcode” section and scan in the plate barcode affixed to the SEP.Place your cursor in the “RNA Elution Plate Barcode” section and scan in the plate barcode affixed to the REP.Click “Assign Plates”.Place your cursor in the “Sample Extraction Plate Barcode” section and scan in the plate barcode affixed to the SEP.Place your cursor in the “RNA Elution Plate Barcode” section and scan in the plate barcode affixed to the REP.Click “Assign Plates”.
This protocol describes the extraction of RNA from samples plated in the lysis buffer (Step 4 above), utilizing magnetic bead nucleic acid purification and using a KingFisher Flex benchtop automated extraction instrument. This protocol also involves scanning barcodes and entering information into the INSPECT application for record-keeping.
Steps involving the INSPECT application are optional and are not essential for performing RNA extraction.
?. [Plate Compression from 96-Well Format to 384-Well Format for RT-qPCR]
Centrifuge:Select one to four (1-4) 96-well REPs and centrifuge  .
This section describes the process of compressing up to four (4) RNA Extraction Plates (REPs) created in Step 7 into one 384-well RNA Working Plate (RWP) that will be used as input for the RT-qPCR detection of SARS-CoV-2, described here:   of each sample is transferred from the REPs to the 384-well RWP.
Centrifuge: 1000 34, 1 min, 4 10
?. [Plate Compression from 96-Well Format to 384-Well Format for RT-qPCR]
Load plates onto epMotion 5075:If necessary, turn on the epMotion 5075 and open the eBlue software on the associated laptop, enter username and password.Load the following epMotion protocol:   Load 1-4 boxes of 50 µL pipette tips into positions A2, A3, A4, and A5, depending on how many REPs are being processed (one box of pipette tips per REP), starting with position A2.Place the REPs into positions B2, B3, B4, and B5, depending on how many REPs are being processed, starting with position B2.Load a clean, barcoded Bio-Rad Hard-Shell 384-well barcoded PCR plate into position C4.Load the following epMotion protocol: Load 1-4 boxes of 50 µL pipette tips into positions A2, A3, A4, and A5, depending on how many REPs are being processed (one box of pipette tips per REP), starting with position A2.Place the REPs into positions B2, B3, B4, and B5, depending on how many REPs are being processed, starting with position B2.Load a clean, barcoded Bio-Rad Hard-Shell 384-well barcoded PCR plate into position C4.
?. [Plate Compression from 96-Well Format to 384-Well Format for RT-qPCR]
Scan plate barcodes into INSPECT application:In the INSPECT app, click the "qRT-PCR" button on the left side:  On the following page, click "Plate Compression":  Set the mouse clicker on the "EpMotion ID" section and scan the blue "EXCITE LAB" label on the EpMotion.Set the mouse clicker on the "First 96-Well Plate and Plate into Array Position B2" and scan the barcode of the REP in position B2.Repeat above step for any other REPs in positions B3, B4, and B5.  Set the mouse clicker on the "RNA Working Plate Barcode" section and scan the barcode on the side of the Bio-Rad Hard-Shell 384-Well PCR plate in position C4.Click "Assign Plates".On the following page, click "Plate Compression":  Set the mouse clicker on the "EpMotion ID" section and scan the blue "EXCITE LAB" label on the EpMotion.Set the mouse clicker on the "First 96-Well Plate and Plate into Array Position B2" and scan the barcode of the REP in position B2.Repeat above step for any other REPs in positions B3, B4, and B5.  Set the mouse clicker on the "RNA Working Plate Barcode" section and scan the barcode on the side of the Bio-Rad Hard-Shell 384-Well PCR plate in position C4.Click "Assign Plates".Set the mouse clicker on the "EpMotion ID" section and scan the blue "EXCITE LAB" label on the EpMotion.Set the mouse clicker on the "First 96-Well Plate and Plate into Array Position B2" and scan the barcode of the REP in position B2.Repeat above step for any other REPs in positions B3, B4, and B5.  Set the mouse clicker on the "RNA Working Plate Barcode" section and scan the barcode on the side of the Bio-Rad Hard-Shell 384-Well PCR plate in position C4.Click "Assign Plates".Set the mouse clicker on the "RNA Working Plate Barcode" section and scan the barcode on the side of the Bio-Rad Hard-Shell 384-Well PCR plate in position C4.Click "Assign Plates".
Steps involving the INSPECT application are optional and are not essential for performing RNA extraction.
?. [MS2 Phage Control Addition]
Set up consumables on the epMotion table:Place a box of 50 µL epTIPs with at least 1 full column of tips into Position A2.Label a clean 10 mL reservoir with “MS2” and current date.Set a reservoir rack into Position B2 of epMotion 5075. Place the "MS2" reservoir into the left-most slot (Position 1) of the reservoir rack. Transfer   of the thawed (1:160 diluted) MS2 phage aliquot into the "MS2" reservoir, using a P1000 pipette and filter tips.Set the SEP plate containing sample and binding beads in Position C2.Set a reservoir rack into Position B2 of epMotion 5075. Place the "MS2" reservoir into the left-most slot (Position 1) of the reservoir rack. Transfer  of the thawed (1:160 diluted) MS2 phage aliquot into the "MS2" reservoir, using a P1000 pipette and filter tips.
1200 µl
"MS2" reservoir can be used throughout the day if multiple plates are being run (create a new plate each morning).
?. [Plate Compression from 96-Well Format to 384-Well Format for RT-qPCR]
Run the program:Remove the seals from the REPs and remove the tip box covers.Click the "Play" button on the top of the screen.Available device will be selected by default - click "Next".Under volume settings, select "Use minimum required volumes."Under worktable settings, leave "Detected tips" selected, but un-select options "Check labware placement" and "Check tube lid removed".Click "Next". Double check that all consumables (plates, tips) are firmly in the correct position and all lids/seals have been removed.Click "Run".Double check that all consumables (plates, tips) are firmly in the correct position and all lids/seals have been removed.Click "Run".
A "Process Control" window will appear, asking for the number of samples present in each of the 96 well REP(s). Follow the instructions below depending on the number of REPs being processed:You will be prompted to enter the number of samples on the first plate; Enter "96" and click "OK"Another window will pop up with the same prompt for the second plate; If compressing only one plate enter "0", other wise enter "96" and click "OK"Another window will pop up with the same prompt for the third plate; If compressing less than 3 plates, enter "0", otherwise enter "96" and click "OK"Another window will pop up with the same prompt for the fourth plate; If compressing less than 4 plates, enter "0", otherwise enter "96" and click "OK"
Once the plates are compressed into the 384-well RWP, it is ready for RT-qPCR detection of SARS-CoV-2. After running the RT-qPCR the RWP can be stored at   .
?. [Sample Extraction Plate (SEP) Preparation]
Set up consumables on the epMotion table:Place a box of 50 µL epTIPs with at least 1 full column of tips into Position A2.Label a clean 10 mL reservoir with "ProtK" and the current date. In the EpMotion hood, add   Proteinase K   into the labelled 10 mL "ProtK" reservoir and place in the left-most slot of the reservoir rack (Position 1).Label a clean KingFisher deep-well 96-well plate with a SEP barcode and place in Position C2.In the EpMotion hood, add  Proteinase K  into the labelled 10 mL "ProtK" reservoir and place in the left-most slot of the reservoir rack (Position 1).
"ProtK" reservoir can be used throughout the day if multiple plates are being run (create a new plate each morning).
1.4 mL
?. [Sample Extraction Plate (SEP) Preparation]
Run the program:Available device will be selected by default - click "Next".Under the volume settings, select "Use required minimum volumes".Under worktable settings, leave "Detected tips" selected but uncheck "Check labware placement" and "Check tube lid removed".Click "Next".Double check that all consumables are firmly in the correct position and all lids have been removed.Click "Run".
?. [Wash and RNA Elution Plate (REP) Preparation for RNA Extraction]
Use epMotion to transfer wash and elution buffers into plates:Load the attached epMotion protocol:  .
?. [Wash and RNA Elution Plate (REP) Preparation for RNA Extraction]
Set up consumables on the epMotion table:Place a box of 1000 uL epTIPs with at least 3 full columns of tips into position A2 of the epMotion.Place a reservoir rack in position B2 with three reservoirs in the three slots as described above in Step 5.Label one clean KingFisher 96-well deep well plate "Wash Buffer" and place into position C2 on the epMotion.Label one clean KingFisher 96-well deep well plate "EtOH" and place into position C3 on the epMotion.Label one clean KingFisher 96-well deep well plate with an "REP" plate barcode and place into position C4 on the epMotion.
?. [Wash and RNA Elution Plate (REP) Preparation for RNA Extraction]
Run the program:Click the "Play" button on the top of the screen.Available device will be selected by default - click "Next".Under volume settings, select "Use minimum required volumes."Under worktable settings, leave "Detected tips" selected, but un-select options "Check labware placement" and "Check tube lid removed".Click "Next".Double check that all consumables (reservoirs, tips, etc.) are firmly in the correct position and all lids have been removed.Click "Run".
Safe stopping point:After plates are filled, seal with transparent plate seals and use a roller press to seal down firmly, OR proceed directly to RNA extraction. The "Wash Buffer" plate will have   wash buffer in each well, the "EtOH" plate will have   EtOH in each well, and the "REP" plate will have   nuclease-free water in each well.
?. [Binding Bead Mix Solution Addition to SEP]
Use the epMotion to transfer binding bead mix solution to SEP:Load the following epMotion protocol:
?. [Binding Bead Mix Solution Addition to SEP]
Run the programClick the "Play" button on the top of the screen.Available device will be selected by default - click "Next".Under volume settings, select "Use minimum required volumes."Under worktable settings, leave "Detected tips" selected, but un-select options "Check labware placement" and "Check tube lid removed".Click "Next".Double check that all consumables (reservoirs, tips, etc.) are firmly in the correct position and all lids have been removed.Click "Run".
After SEP plate is filled, proceed to MS2 phage control addition (step 6 below) immediately.
?. [Binding Bead Mix Solution Addition to SEP]
Set up consumables on the epMotion table:Place a box of 1000 uL epTIPs with at least 1 full column of tips into Position A2 on the epMotion.Place a reservoir rack in Position B2, and add the    reservoir with binding bead solution to the left-most slot (Position 1).Place the SEP containing samples into Position C2.Place a reservoir rack in Position B2, and add the   reservoir with binding bead solution to the left-most slot (Position 1).
100 mL
?. [MS2 Phage Control Addition]
Run the program:Click the "Play" button on the top of the screen.Available device will be selected by default - click "Next".Under volume settings, select "Use minimum required volumes."Under worktable settings, leave "Detected tips" selected, but un-select options "Check labware placement" and "Check tube lid removed".Click "Next".Double check that all consumables (reservoirs, tips, etc.) are firmly in the correct position and all lids have been removed.Click "Run".
?. [Viral RNA Extraction using KingFisher Flex]
Extract RNA using the KingFisher Flex:Turn on the KingFisher Flex (power button located on the left side of the instrument at the bottom). Push the right arrow to enter User folder (second tab), scroll down to "RNA", click "OK" button, and select the following protocol:   Click "Start".Push the right arrow to enter User folder (second tab), scroll down to "RNA", click "OK" button, and select the following protocol:
?. [Viral RNA Extraction using KingFisher Flex]
The instrument will prompt you to load the following plates in order:Tip Comb Plate: there should be a clean, 96-well plate in this position, place a fresh ‘KingFisher 96 Tip Comb for DW Magnets’ into the plate so that each plastic comb fits into each well in the plate. Hit “START”.Elution Plate: load the REP, ensuring well A1 is next to the A1 designation in upper-left-hand corner.Hit "START".80 Ethanol: load the EtOH plate.Hit "START".Wash Buffer Plate: load the Wash plate.Hit “START”.Sample Plate: load the SEP, ensuring well A1 is next to the A1 designation in upper-left-hand corner.Hit “START”.The instrument will automatically begin to process samples and list a countdown timer in minutes in the lower right-hand corner of the screen.
?. [Viral RNA Extraction using KingFisher Flex]
When finished, the instrument will prompt you to take out the REP. Remove used consumables from Flex:Hit "START".Remove the SEP and place to the side.Hit “START”.Remove the Wash plate and place to the side.Hit “START”.Remove the EtOH plate and place to the side.
Safe Stopping Point:The RNA Elution Plate (REP) now contains purified RNA from the samples. Each well should contain   . Seal REP with an AlumaSeal, using a plate roller to ensure tight adherence.If proceeding to the "Semi-Automated and Miniaturized Detection of SARS-CoV-2 using the TaqPath COVID-19 Multiplex Real-Time RT-PCR Assay" protocol, proceed to Step 8 to compress samples into 384-well plate format. This compression process allows for up to four (4) REPs to be compressed into one 384-well RNA Working Plate (RWP); if more than one plate is being processed, place finished REPs in the fridge at   , while waiting for the rest to finish, and then proceed with all REPs to Step 8.
Pour the liquid from the SEP, Wash, and EtOH plates into a labelled chemical waste container, then discard the plates into an appropriate waste container.
?. [Sample Collection]
Anterior nares swabs are obtained via self-collection.Individuals are given an individually wrapped, sterilized swab (Nantong Renon Laboratory Equipment, PN: NLD602-1) and instructed to:Insert the swab approximately 1.5 cm into the nostril, Twist the swab inside the nostril three (3) times, and Hold the swab against the septum for a total of 15 seconds, thenRepeat these steps on the other nostril with the same swab.The swab is then inserted into a into a 5 mL sample tube containing  molecular transport media (MTM), Mawi preservation solution, or 5% sodium dodecyl sulfate (SDS) (Table 1). If required (depending on the length of the swab handle), the handle of the swab is broken off at a pre-scored area directly above the tip so that the tip remains in the solution. MTM, Mawi, and SDS are able to inactivate the SARS-CoV-2 virus while preserving the viral RNA needed for detection.Table 1. Acceptable sample storage temperatures and durations.Specimen TypePreservation SolutionStability and StorageAnterior Nares SwabMawiStore at Room Temperature up to 1 month. Store at -90 C to -70 C indefinitely.Anterior Nares SwabMTMStore at Room Temperature up to 7 days. Store in Refrigerator (2 C to 8 C) up to 28 days. Store at -90 C to -70 C indefinitely.Anterior Nares SwabSDS (5%)Store at Room Temperature up to 1 year. 5% concentration.
1.5 mL
Specimen TypePreservation SolutionStability and StorageAnterior Nares SwabMawiStore at Room Temperature up to 1 month. Store at -90 C to -70 C indefinitely.Anterior Nares SwabMTMStore at Room Temperature up to 7 days. Store in Refrigerator (2 C to 8 C) up to 28 days. Store at -90 C to -70 C indefinitely.Anterior Nares SwabSDS (5%)Store at Room Temperature up to 1 year. 5% concentration.
This protocol includes steps to follow to track patient data using the INSPECT application, a sample data tracking tool developed for SARS-CoV-2 testing. These steps are not essential to the RNA extraction process and are noted as such.
?. [Sample Accessioning and Plating using Hamilton STAR]
Set up the Hamilton STAR:Wipe down the sample carriers and Hamilton deck with 70% EtOH.On the computer desktop associated with the Hamilton STAR, open "Sample Transfer".In the pop-up window, enter the number of samples and controls to be plated (max 480).Select the "Sample Source Type".    If running only MTM samples, choose "bottom barcode".    If running only Mawi samples, choose "round barcode".    If running another type or a combination of sample types, choose "round bottom".Enter your initials in capital letters.Hit "Continue".
Retrieve one Sample Extraction Plate (SEP) from Step 2 containing   proteinase K per well for each batch of 94 samples to be processed (maximum of 5 batches per Hamilton run). Ensure that the plate has a barcode sticker placed on the right side of the plate, centered at the upper right edge of the skirt.
NOTE: The "round barcode" designation is used for 5 mL tubes with barcode labels added to the side of the tube (these tubes have a round bottom). The "bottom barcode" designation is used for Thermo bottom-barcoded tubes (these tubes have a flat bottom). See Step 1 for more tube details.
?. [Sample Accessioning and Plating using Hamilton STAR]
Load the carriers accordingly, checking off each box on the screen in order:Load tip carriers: Ensure sufficient  filter tips (1000 µL) are loaded. To fill each 96-well SEP, 96 tips are needed (94 samples and 1 negative and 1 positive control tube each containing 1 mL collection media only).Load sample carriers: Load the 32-position Sample Carriers with the sample tubes collected in Step 1, starting from the carrier that will be placed at the far right of the Hamilton deck (Deck Position 34), filling from the top to the bottom (Carrier Positions 1 through 32), then moving to the next carrier (Deck Position 33), filling from the top to the bottom (Carrier Positions 1 through 32), and so on. [See note below for more details.]Load plate carrier: Load the appropriate number of SEPs starting with the cavity furthest toward the back and double-check that the SEP barcode sticker is attached to the right face of the plate towards the top. Make sure that well A1 for each SEP is located in the upper left corner.Once the autoload trays are loaded, click "Continue".
Begin by inserting barcoded, blank tubes (tubes containing collection   collection media but no patient swab sample) into the carrier positions that will go to SEP well H3 (positive control) and A4 (negative control). This translates to tube positions 24 and 25 of the first of the three racks for each single SEP. For one SEP plate this is deck position 34, for 2 SEP plates this is deck positions 34 and 31, for 3 plates this is deck positions 34, 31 and 28, for 4 plates this is deck positions 34, 31, 28, and 25, for 5 plates this is deck positions 34, 31, 28, 25, and 22.Insert sample tubes to the remaining positions on the carriers, starting with the right-most carrier (Deck Position 34), and moving to the left once full.Decap tubes (either manually or using an assisted tube decapper) and ensure that tube barcodes are facing the opening on the right side of the racks so they can be scanned by the Hamilton.
?. [Sample Accessioning and Plating using Hamilton STAR]
Run the program:A screen will pop up to determine where in the first carrier the tips are loaded: drag and drop to highlight where tips are loaded (gray = no tip, brown = tip).Click "OK".The Hamilton will pull in all carriers, if any barcodes are not read, the instrument will return an error. If this happens, check to make sure the barcode is facing the window on the right side of the carrier, then click “Repeat” → “Execute”.Once no errors are found, the instrument will transfer  from each sample tube into the loaded SEP(s).
200 µl
?. [Sample Accessioning and Plating using Hamilton STAR]
Upload the plate map data generated by the Hamilton into the INSPECT application:On the Hamilton desktop computer, find the most recent .csv file created during this sample transfer. This file will list the sample barcode and the well in the SEP that each sample was added to. This is the "Platemap File".In the INSPECT app, click the "qRT-PCR" button on the left side:  On the following page, click "Submit Platemap".  Click "Choose File" to upload the Platemap file from the Hamilton. To enter the ID of the Hamilton machine used, use a barcode scanner to scan the Hamilton ID blue sticker on the right-hand side of the instrument.Type your initials into the "Name of Person Uploading Platemap" field.Click "Submit".On the following page, click "Submit Platemap".  Click "Choose File" to upload the Platemap file from the Hamilton. To enter the ID of the Hamilton machine used, use a barcode scanner to scan the Hamilton ID blue sticker on the right-hand side of the instrument.Type your initials into the "Name of Person Uploading Platemap" field.Click "Submit".Click "Choose File" to upload the Platemap file from the Hamilton. To enter the ID of the Hamilton machine used, use a barcode scanner to scan the Hamilton ID blue sticker on the right-hand side of the instrument.Type your initials into the "Name of Person Uploading Platemap" field.Click "Submit".To enter the ID of the Hamilton machine used, use a barcode scanner to scan the Hamilton ID blue sticker on the right-hand side of the instrument.Type your initials into the "Name of Person Uploading Platemap" field.Click "Submit".
Steps involving the INSPECT application are optional and are not essential for performing RNA extraction.
?. [Sample Accessioning and Plating using Hamilton STAR]
Once Hamilton run is finished:When sample transfer is complete, Hamilton will display a "Method Complete" sign.Pull out the carrier tray and remove the SEP(s). Seal SEP with aluminum plate seal, using a plate roller to ensure a tight seal.Recap each of the 94 sample tubes with a Globe Scientific Polyethylene (PE) Stopper Flange Cap (PN: 118127R, or equivalent)Place each set of capped 94 sample tubes into a 4" freezer storage box and leave at room temperature for up to one week.Wipe down sample carriers and Hamilton deck with 70% EtOH.
Sample tubes can be discarded once the original samples are no longer needed.