Operating the MultiProbe (made by Packard, serviced by PerkinElmer)
(Last updated: March 2020)
*LEGACY PROTOCOL* (MultiProbe replaced with Janus in ~ 2021)
Making cherry pick robot files in Excel:
1) Use past files as examples for how a robot file should be organized.
2) The file uses the following headers:
- Samp_ID: order of entries, numbered from “1” onwards consecutively.
- Asp_P_ID: plate ID number (“Plate1_001, Plate2_001”) within the MultiProbe file. A plate that appears multiple times in the same Excel sheet should have the same plate ID number.
- Sour_Well: the source well for the sample.
- Sour_Plate: the name of the plate containing the sample.
- Dsp_P_ID: the new plate in which all the samples will be deposited. This is usually just named “Dest_Plate.”
- Dsp_Well: the well in which each sample will be deposited on the destination plate.
- Pk_vol: volume in uL that will be transferred from one plate to another.
3) Save the file in a .csv format, then upload it onto the thumb drive and use that to transfer the file to the MultiProbe computer.
Operating the MultiProbe:
1) Open “Cherry-Pick…Kasia.MPT” on the MultiProbe computer’s desktop.
2) Double-click the first user file from the list on the left, or create a new protocol by clicking the icon of a globe.
3) Click the “Run Time Parameters” tab, then hit “Files” and “Browse.” Select your file from the thumb drive, then hit “Open.” You should see a copy of the Excel file you uploaded in the window at the bottom of the screen. Check the entries to make sure the file is accurate.
- The generated robot file doesn’t always work with the ancient MultiProbe computer. If there is a formatting issue (every entry is in a single line running across the window), check to see if the robot file is in the correct .csv format. If that fails, use the file “Working file for MultiProbe 2” as the template.
- Open the template file and delete all data within except the headers.
- Copy/paste the data from the correct robot file into the template file.
- Hit “save.” This should solve any issues with the MultiProbe program.
4) Place the rePCR plates you have thawed, vortexed (30 sec), and spun down (1 min) in the correct locations. The positions on the deck number 1-20 in a column fashion (the top right-hand rack is 1, the one right below that is 2, and so on).
- Check the order of the plates against the QC checklist generated by the cherry pick function on Rong’s desktop.
5) Place a box of blue PerkinElmer RoboRack 200uL Clear Non-Conductive Tips in the “tips” position.
6) Label a new 96-well U-bottom plate with the order #, the description (“cherry pick” in this case), and the date. Place it in the position right below the tips that is labeled “Destination Plate.”
- The destination plate (“Dest_Plate”) should be a 96-well U-bottom due to the settings of the MultiProbe.
7) Click “Execute Test.” If necessary, check the location of each position by clicking “Plate locations” and visually doing a check. Otherwise, hit “Reset Tip Box” to make sure the number of tips in the program matches the actual number of tips in the box. Then hit “OK.”
8) After the tips window closes out, hit “Next,” then hit “Start.”
9) Monitor the MultiProbe for a few moments to make sure it is executing the protocol correctly.
- Also monitor the tip box. The MultiProbe will prompt you for a new box of tips if it runs out.
- If at any time you need to stop the execution, hit “Pause” on the screen. In emergencies, hit the giant red button located on the bottom on the MultiProbe.
10) After cherry-picking samples, use the PlateLoc to seal all the rePCR plates and put them back into their correct positions in the racks in the -20 freezer. Seal the labeled cherry pick plate as well and store it on the door of the fridge if it’s not immediately being used for PCR.
11) If working on the IVT pipeline, move onto PCR.
Normalization
We use the MultiProbe for normalizing plates, which means ensuring that each well on the plate has the same concentration (and with our workflow, typically results in each well also having the same volume). This step is done towards the end of the IVT pipeline. In addition, colleagues may ask for assistance with normalizing their own samples and plates (such as 96-well plates of plasmid constructs).
Making normalization files in Excel:
1) Determine the concentration of each sample using the Nanodrop, then use that number and the desired final concentration to work out what the final volume needs to be.
- Use the C1V1=C2V2 equation to determine how much water needs to be added to each sample by subtracting out the volume of sample needed from the desired final volumn.
- The MultiProbe cannot pick up less than 2uL of liquid, so some samples may need to be normalized by hand. When creating the normalization file, make sure none of the samples need less than 2uL of either the sample or water for normalization. Sort the spreadsheet by sample concentration to check.
2) Enter the amount of liquid needed from each plate into a template modeled after the cherry pick robot files (see “Making cherry pick robot files in Excel”). Each well should have its own row (and therefore its own entry number/Samp_ID).
3) Do the same for the amount of water needed for each sample. Make sure that the amount of water needed for normalization has the same destination well as its corresponding sample.
4) Set up the MultiProbe .csv file using a file for cherry picks as a template.
5) Enter each sample and water needed per well as a separate row. For instance, if both sample and water needs to be added to well A01, there should be two rows in the Excel spreadsheet with A01 listed as the Dsp_Well.
- Water can be set up on the MultiProbe deck in two ways.
- Fill a clean, spare 96-well U-bottom plate with deionized water. In this case, the Sour_Well on the spreadsheet should be set up so that water is pulled from each corresponding well.
- Use a water reservoir. Take an empty tip container, wipe it clean with 70% ethanol, and let it air-dry. After the container is dry, fill it with deionized or nuclease-free water. In this case, the Sour_Well on the spreadsheet for the water entries matters little.
- In the Asp_P_ID column, list the water plate after the sample plates.
- The name listed under Sour_Plate doesn’t matter for this protocol; it is purely for reference.
Using the MultiProbe for normalization:
1) Set up the plates (and water reservoir if you have one) on the MultiProbe deck as you would for a cherry pick. Make sure tips are in the correct location and the number of tips match what is in the MultiProbe program.
- We generally normalize samples with nuclease-free water (CAS# 7732-18-5, US Biological Life Sciences), but sometimes there will be special requests to normalize using another solution, such as elution buffer.
2) If using a water reservoir, the labware in the MultiProbe program may have to be changed, as the Plate1_001 location is usually programmed in as 96-well u-bottom plate. To change the labware, go to the main screen of the MultiProbe program.
- Right-click on the Plate1_001 location and select “Remove labware from deck.”
- Right-click again on the newly empty spot and select “Add labware…”
Category: reservoir
Labware: water reservoir
Support tile: Plate-Adapter support tile
Name: water reservoir
To switch the labware back to Plate1_001, follow the same steps to remove the labware already in that location and to add a new piece of labware. Use the following specifications:
Category: 96 well plate
Labware: 96 well round bottom (NUNC)
Support tile: Plate-Adapter support tile
Name: Plate1
The type of labware will change, depending on what kind of plate is being used:
- 96-well sample plate: 96 well round bottom (NUNC), deep maroon on the deck layout.
- PCR Plate: v-bottom, gray on the deck layout.
- Deep-well Plate: DRSC Source, light pink on the deck layout