ACRF EpMotion programming SOP Created on 11/11/2009 11:49:00 AM

ACRF EPmotionprogramming SOP

1 Start up procedure

2 Virtual Setup

Composed / Cairo Forrest / Research Assistant
Reviewed / Pavel Bitter / ACRF Manager
Version / 1.0
Date / 11.11.2009

3 Programming the epMotion

4Physical Setup

5Running the program

1 Start up procedure

-If you are part of the cancer group use the ‘cancer’ robot otherwise use the ‘All Groups’ EPmotion

-Start up the computer / instrument by following the instruction sheet on the wall above the computer:

1) Turn on EpMotion (green switch at bottom left of robot)

2) Turn on the power board behind the monitor (red switch on power board)

3) When computer has booted Press ‘OK’ for the first login, then on 2nd log in screen enter password (the password can be found on a sticky label on the monitor)

4) Now wait approximately 3 minutes before login into the “epBlue” software for the EpMotion (until you see the robot has adjusted and homed the robot arm). Use the password and username, displayed on the label on the monitor for the login.

-Open the ‘epBlue client’ software from the desktop.

-The software directs you to the ‘home’screen

-Go to the ‘Tasks’ tab (lower part of the screen) and choose ‘create / edit applications’, in the popup window “Create new application”go to “User”

-under ‘user’ choose ‘ACRF’ and under ‘folder’ make your own folder with your name

-under ‘application’ make a new application by clicking on the icon of a blank sheet (hovering with the mouse over it will show “create new application).

-a new popup window will appear “Create a new application”

-name the new application naming with the “date and name” of your choice second (eg; 091109_testrun)

-under “Device type…. Please select” choose “epMotion” and click on “Create”

-You are then taken to the WORK program screen (This is the second tab on the right vertical menu bar: HOME, WORK, LABWARE, FUNCTIONS ADMIN).

In the ‘WORK’ tabthere are 4 new vertical tabs which are 1. ‘Worktable’ 2) ‘Procedure’ 3) ‘Run’ and 4) ‘Control’

2 Virtual Setup

-The programming begins in ‘Worktable’ you must select the labware you want to place on the deck, this will be a virtual representation of the hardware you will then later physically place in the exact positions on the epMotion robot.

-Under ‘labware’choose:

1)TIPS

In‘Labware-type’click on “TIPS” and in‘Sub-type’ choose ‘Tips’then in the‘Labware’ tab click and drag with the mouse the tips labelled ‘tip50 epTIPS Motion 50ul’ icon up to the ‘Worktable’ display and place it in your chosen square space provided (either in the A1 position or the A2 position)

2)MASTER MIX

In ‘Labware-type’click on‘Equipped Racks and Modules’ and then in‘Sub-type’ choose ‘Equipped Racks and Modules’. In‘labware’click and drag with the mouse the 24 well rack labelled ‘EP_Rack_1_5ml_X Eppendorf Thermorack for 24xSafe’ icon up to the ‘Worktable’ display and place it in your chosen square space (name this labware file as MasterMix or MM when prompted to do so)

3)ADAPTER

Note: adaptors are used to speed up pipetting of the robot as the height is the same for tips, samples and cDNA. They are not needed and if not programmed, the program will still work, but if they are programmed they MUST be placed under the thermoplates. The 5070EPmotions (”cancer” and “all groups”) have 55mm adaptors, the 5075EPmotion (mouse gneotyoping) has 40mm adaptors.

In ‘Labware-type’click on‘Adapter’. Then in‘Sub-type’ choose ‘Height’. In ‘labware’ click and drag with the mouse the adapter labeled ‘Adap-55 Height adapter 55mm’icon up to the ‘Worktable’ display and place it in your chosen square space (use a height adapter for your 384 well plate and a height adaptor for your 96 well plate)

4)THERMOBLOCKS With PLATES

Note: ALWAYS use thermoblocks with plates to place a plate on the deck, there are no plates alone, they always go with the thermoblocks, which hold them precisely where they need to be, so you cannot select a plate only, hence you will find nothing to choose from in the category plates…

In ‘Labware-type’click on‘Thermoblocks with Plates’. And then in‘Sub-type’ choose ‘Thermoblocks with Plates’. In ‘labware’ click and drag with the mouse the384 well thermoblock with plate labelled ‘ABI-Thermo_40-1 Biosystems 384 Well PCR plate’ icon up to the ‘Worktable’ display and place it on top of one of the two height adapters (if you have programmed them). When the naming window pops up give them sensible names (for example:‘PCR plate’ or “destination plate”. Then click and drag the 96 well thermoblock with plate labelled ‘BIOR_PCR_300_1 Bio_Rad’ onto the remaining adapter and finally name this as ‘cDNA’or “samples” when prompted to do so.

Note: The PARK1, PARK2 and PARK3 positions are virtual spaces for storing extra samples or master mixes that you might want to also substitute into the active positions at some point. If you position “thermoblocks with plates” it means that in the program you can later exchange any given position with the PARK1,2, 3 position, but when the program is actually running on the EPmotion you will have to come to the instrument and exchange the thermoblocks with plates manually.

3 Programming the epMotion

-Still within the ‘Work’ tab---Go to the next vertical tab down under ‘Worktable’ called ‘Procedure’ which is where you will create a specific program (set of instructions for the robot).

-In the ‘Commands’ box (lower icon selection box) you will see command icons. The first is ‘Number of Samples’, next is ‘Sample Transfer’ and the next is ‘Reagent Transfer’and “Exchange” (these are the three commands you will use the most). The command “Sample Transfer’is used for one-on-one sample transfer from for example your cDNA to the PCR plate, each time using a new tip. Thecommand ‘Reagent Transfer’is used to aliquot the Mastermix out from one well into many with the same tip.

Note: If you are doing a very long program you can utilize the ‘Comment’ icon to break up sections of the program by placing a worded comment in front of the section. This can make it easier to back track through a long program and look for certain sections of the program that may need to be altered. The “Comment” command will not affect the actual program, its only cosmetic in the program structure.

Note: You can also utilize the ‘Exchange’ icon to pause the machine so that you can add, remove or swap samples/ labware in the middle of a run if need be. The robot will pause when it comes to the “Exchange” command and wait until you press “return” on the keyboard of the epMotion when the program is running.

To program you must click on a icon and drag it into the ‘Procedure’field. You need to always put a ‘Number of Samples’ command before a ‘Sample Transfer’ or a ‘Reagent Transfer’ command, that tells the robot how many times you wish the following command to be executed.

  1. ‘Number of Samples’

Click on ‘Number of Samples’ and drag it up into the ‘Procedure’ field. Drop it before the ‘End of method’command line which will already be there. On the bottom left hand side of the screen under the ‘Number of Samples’ field tick the ‘Fixed number of samples’ box and then below it in the field provided type in the number of samples you want to transfer.

  1. ‘Sample transfer’(for example to transfer cDNA from the 96 well plate to the 384 well PCR plate)

Click on the ‘Sample transfer’ icon and drag it up to drop it under the ‘Number of Samples’ icon you placed previously. Now you will notice on the bottom left hand side of the screen that there is a ‘Sample Transfer’ field with details to be filled out. There are 4 horrizontal tabs below this (‘Parameter’/ ‘Options’/ ‘Mix’ / ‘Liquid Type’). Within ‘Parameter’ (which is where you will initially be) fill in all the fields required

1) Don’t change the ‘Pipet Tool’, leave as “TS_50”

Note: Don’t tick “Filter tips” as we do not have filtered tips

Note: There is a 8 channel tool which can be used. Then select “TM_50_8”. Be aware that then you will have to program a stop in the program to exchange the tool later for the TS_50 for pipetting Mastermixes.

2) Choose a‘Volume’setting (for example 1.0ul for cDNA transfer into a 10ul Taqman reaction)

Note: you can choose 1.5ul for example or 2.2ul. The pipetting with comma values is not guaranteed to be accurate but it will be approximate, values lower than 1ul will not be accepted by the program and lead to an error.

3) Don’t change the ‘Transfer type’ as “pipette”(This means it will take a new pipette tip for each sample and not contaminate)

4) In ‘Source’choose the name of your source plate (for example ‘cDNA’) from your choices

5) In ‘destination’choose the name of your destination plate (for example ‘PCR’).

6) Click on the ‘Pattern’ tab (still within the ‘Parameters’ field) and click on which sample from source plate you want to transfer into which well of the destination plate. (You can placeone source sample in multiple spots in the destination plate).

The patterning in the EPmotion is very intuitive and saves a lot of work. It will think logically and fill in the empty wells in a predictable pattern once you set the initial few samples to “explain” your pattern.

After your first sample, the next sample will define the pattern, whether it goes from left to right or from top to bottom is defined by the first two samples. Even more complex patterns are possible, like every third or fourth well etc. try it out.

Make some sample transfers and then press the ‘OK’ button (on the bottom right) and then click on ‘Pattern’ again, the software will have automatically filled the empty wells according on how many samples you have given in the previous step (“Number of Samples”).

Note: There are 2 boxes below for ‘irregular source pattern’ and ‘irregular destination pattern’ which can be used if you are taking samples in an irregular pattern from your 96 well plate or putting them in an irregular pattern in your 384 well plate (ie; an irregular pattern is not in rows or lines or anything that is too complex for the patterning skills of the program. The downside of irregular patterning is that you will have to program every single transfer manually, the pattern will not be filled automatically as the program does not follow a regular patterning.)

7) Finally, go to the ‘Options’ tab at the bottom of the screen (one to the right of the ‘Parameters’ tab where you currently are and you will bring up the ‘Options’ field which has a series of boxes that are unticked.. Tick the ‘Aspirate from the bottom’ box.This will force the robot to take the liquid from the bottom of the well. If you leave as is the default, you will have to enter very accurately the amount of Mastermix you have in each tube, because the robot will calculate where the top of the liquid is and only plunge that deep into the well. If you force the robot to pick up its liquid from the bottom, you do not have to be precise in giving the volume of your mastermixes later.

  1. ‘Reagent transfer’ (for example to transfer Master Mix from the 24 well rack to the 384 well PCR plate)
    You must click and drag another ‘Number of Samples’ icon into the ‘Procedure’ field first. Then click ‘Fixed Number of Samples’ BUT unlike for the ‘Sample Transfer’here the number of samples means how many times each Master Mix will be dispensed into the destination plate (rather than the number of Master Mix samples that will be dispensed)

1)Then click on the ‘Reagent Transfer’ icon and drag and drop it next in the ‘procedure’ field. In the bottom right hand corner of the screen a box much like that which appeared for ‘Sample Transfer’ will appear but named ‘Reagent transfer’ with the same 4 tabs below in a horizontal arrangement (‘Parameter’ / ‘Options’ / ‘Mix’ / ‘Liquid type’). Fill these details out much in the same way as you did for the ‘Sample transfer’.

1) Don’t change the ‘Pipet Tool’, leave as “TS_50”

Note: Don’t tick “Filter tips” as we do not have filtered tips

Note: There is a 8 channel tool which can be used. Then select “TM_50_8”. Be aware that then you will have to program a stop in the program to exchange the tool later for the TS_50 for pipetting Mastermixes.

2) Choose a ‘Volume’ setting (for example 9.0ul for Mastermix into a 10ul Taqman reaction)

Note: you can choose 1.5ul for example or 2.2ul. The pipetting with comma values is not guaranteed to be accurate but it will be approximate, values lower than 1ul will not be accepted by the program and lead to an error.

3) Don’t change the ‘Transfer type’ as “multidispense”(this means that one Master Mix can be dispensed into multiple wells without changing tip each time which would waste tips and time)

4) In ‘Source’ choose the name of your source plate (for example ‘Mastermix’) from your choices

5) In ‘destination’ choose the name of your destination plate (for example ‘PCR’).

6) Click on the ‘Pattern’ tab (still within the ‘Parameters’ field) and click on which sample from source plate you want to transfer into which well of the destination plate. (You can placeone source sample in multiple spots in the destination plate).

After your first sample, the next sample will define the pattern, whether it goes from left to right or from top to bottom is defined by the first two samples. Even more complex patterns are possible, like every third or fourth well etc. try it out.

Make some sample transfers and then press the ‘OK’ button (on the bottom right) and then click on ‘Pattern’ again, the software will have automatically filled the empty wells according on how many samples you have given in the previous step.

Note: For the next Master Mix in the rack you must program another ‘Number of Samples’ icon and fill in the details and another ‘Reagent Transfer’ icon and again fill in the specific details likewise so that every Master Mix you want to transfer has its own ‘Number of Samples’ and ‘Reagent Transfer’ icon!

7) Go to the ‘Options’ tab as we did for ‘Sample transfer’ and select ‘Aspirate from the bottom’

Note: If you find that bubbles are forming in the pipetting of your Master Mix you can go into ‘Liquid type’ within the ‘Reagent Transfer’window and use another setting which may increase the time of aspiration and/or dispensing to avoid bubbles especially if you have a very viscous liquid you are pipetting (using the Glycerol setting can work for viscous master mixes such as ABI TaqMan Power SYBR master mix.

Note: the best way to program is to copy and paste chunks of commands and just adjust the details (ie; the pattern of source and destination wells as this eliminates the need to go into ‘options’ each time and select the ‘Aspirate from the bottom’options each time as this info will be transferred in the copy and paste!)

Finally save the program with the “Save” button (it will not start without saving) and press the little “tick-CMD-button” it checks the programfor errors. If any are found, go back to the line indicated and correct the wrong program step.

4Physical Setup

-To run the Now you must physically set up the EpMotion robot’s platform to mimic the virtual set up you have created within ‘Worktable’ by putting the physical labware in the corresponding positions.

-The tools are kept in their original cardboard box behind the robots.The tool must be -manually attached prior to usage by attaching and gently locking it in with the leaver as shown to you in training.

-The labware for both (‘all groups’ and ‘cancer’) robots are located in the fridge in the PrePCr room on the bottom shelf and are labeled either ‘all groups’ or ‘cancer’.

-The height adapters are located next to the robots.

-the tips can be purchased from ACRF facility

-Place the tip box in position, take off the lid

-Place the 24 well master mix rack in (with 1.5ml eppendorf tubes containing your master mix in an ordered fashion and the lids open and pointing in the same direction so the throat is held by the groove)

-Place the two height adapters in the vacant spaces (if they have been programmed

-Place the 96 well thermoblock on top of one height adapter and the 384 well thermoblock on top of the other. Then place a 96 well Biorad plate (containing your cDNA) and a 384 well Roche plate on top of their respective thermoblocks (both these plates and the EpMotion tips are stocked in the ACRF and can be purchased anytime).