DNA Extraction/Purification and RNA Purification

I have written three protocols for extracting and purifying DNA and RNA. The DNA Extraction & Purification protocol follows steps 6-18 of the Omega Bio-tek Mag-Bind® Blood & Tissue DNA HDQ 96 Kit Quick Guide for DNA extraction and purification from up to 10 mg tissue. The RNA Purification protocol follows the steps given in part III of the Zymo Research Quick-RNA™ MagBead protocol. The DNA & RNA Extraction protocol follows the steps given in part IV of the Zymo Research Quick-DNA/RNA™ MagBead protocol.

All three protocols are very similar, so to avoid redundancy I've grouped them together here.

The wells containing RNA samples are specified in the code and can be changed manually or pasted from a spreadsheet.

Note that columns must be filled in order, and one column must be full before moving on to the next one.

sample_wells_data = """
        1   2       3       4       5       6       7       8       9       10      11      12
A   TRUE    FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
B   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
C   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
D   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
E   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
F   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
G   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
H   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE   FALSE
"""
sample_wells_data = sample_wells_data.replace('\t', ',')

These protocols use both the 8-channel and single-channel pipettes, so there are three parameters for starting tips: multi_starting_col, single_starting_row, and single_starting_col. Since the 8-channel pipette picks up an entire column of tips at a time, it is best to use separate tip racks for the pipettes. The starting row for the 8-channel pipette tips is always row A, so we don't need a parameter for that.

The run function for all three protocols starts by reading the data given for the sample well plate. selected_wells is a list of every well that contains a sample.

csv_wells_data = sample_wells_data.splitlines()[1:]
wells_data_reader = csv.DictReader(csv_wells_data)
selected_wells = []
for row in wells_data_reader:
    r = row[""]
    for i in range(1,13):
        new_key = r + str(i)
        row[new_key] = row.pop(f'{i}')
    del row[""]
    keys = list(row.keys())
    true_keys = []
    for key in keys:
        if row[key] == "TRUE":
            true_keys.append(key)
    selected_wells.extend(true_keys)

Then the number of columns in the plate that contain samples, cols, is calculated by integer division of the number of wells used by 8 (the number of wells in a column).

cols = math.ceil(len(selected_wells) / 8)

The last thing to do with the wells data is to create sample_wells, which is a list of length cols in which every item is itself a list representing one column of the well plate.

sample_wells = []
for i in range(cols):
    start_index = 8 * i
    if i == cols-1:
        sample_wells.insert(i, selected_wells[start_index:])
    else:
        sample_wells.insert(i, selected_wells[start_index:start_index+8])

Aside from the standard labware on the deck (which differs slightly for each protocol), the three protocols all also load a magnetic plate in the off-deck* location. The protocols pause at various points for the user to move this plate on and off the deck.

For DNA Extraction/Purification and Total RNA Purification, this block is defined as follows:

magnetic_block = protocol.load_labware("96well_plate_2000ul_on_magnet_plate", protocol_api.OFF_DECK)

For DNA/RNA Purification, two separate blocks are defined:

dna_magnet_plate = protocol.load_labware("96well_plate_2000ul_on_magnet_plate", protocol_api.OFF_DECK)
rna_magnet_plate = protocol.load_labware("96well_plate_2000ul_on_magnet_plate", protocol_api.OFF_DECK)

Even though the same magnet plate is used for both the DNA and RNA well plates, the protocol is written in this way so that the robot recognizes the difference between a well plate and a well plate on top of the magnetic block.

Because many similar steps are performed throughout the three protocols, we utilize some custom functions. The first of these is add_and_mix.

For DNA Extraction/Purification add_and_mix takes in two parameters: vol, the volume to add to the samples, and source, a list of wells that contain the liquid to be added. This list can contain just one well, but when dealing with greater numbers of samples, it is possible that multiple reservoir wells will have to contain the same liquid to account for the amount needed. The function automatically determines which well to aspirate from if this is the case.

As its name would suggest, add_and_mix adds the specified volume of specified liquid to each of the samples and mixes the solution by pipetting up and down repeatedly.

For Total RNA Purification and DNA/RNA Purification, the function has an additional parameter, mix_vol, which indicates the volume to pipette up and down when mixing. By default, mix_vol is 250µl. For the DNA/RNA Purification protocol, the function has a fourth parameter, dest, which indicates which sample plate to add the solution to.

def add_and_mix(vol: float, source: list[protocol_api.Well], dest: protocol_api.Labware, mix_vol=250.0):
    for i in range(cols):
        src = source[i // (12 // len(source))]
        if i == cols-1:
            for well in sample_wells[i]:
                left_pipette.transfer(volume=vol, source=src, dest=dest[well], blow_out=True, blowout_location="destination well", new_tip="always", mix_after=(10, mix_vol))
        else:
            loc = "A" + str(i+1)
            right_pipette.transfer(volume=vol, source=src, dest=dest[loc], blow_out=True, blowout_location="destination well", new_tip="always", mix_after=(10, mix_vol))

The second function is aspirate_supernatant, which aspirates and discards the supernatant in each well after the magnetic particles have been cleared from the solution.

For DNA Extraction/Purification and Total RNA Purification, aspirate_supernatant has just one parameter, num_aspirations, to determine how many times to perform the process.

In the DNA/RNA Purification protocol, aspirate_supernatant has two more parameters, source and dest. source indicates which sample well to aspirate from, and dest indicates where the aspirated supernatant should be dispensed.

def aspirate_supernatant(num_aspirations: int, source: protocol_api.Labware, dest: protocol_api.Labware):
    for i in range(cols):
        if i == cols-1:
            for well in sample_wells[i]:
                left_pipette.pick_up_tip()
                for j in range(num_aspirations):
                    left_pipette.aspirate(location=source[well].bottom(z=-1), volume=250)
                    left_pipette.dispense(location=dest[well].bottom(z=5))
                left_pipette.drop_tip()
        else:
            loc = "A" + str(i+1)
            right_pipette.pick_up_tip()
            for j in range(num_aspirations):
                right_pipette.aspirate(location=source[loc].bottom(z=-1), volume=250)
                right_pipette.dispense(location=dest[loc].bottom(z=5))
            right_pipette.drop_tip()

These are the only functions needed for the DNA Extraction/Purification protocol, but the Total RNA Purification and DNA/RNA Extraction protocols have a third function, add_mix_pellet_aspirate. This function calls add_and_mix, transfers the sample plate to the magnetic block, calls aspirate supernatant, and then removes the sample plate from the magnetic block.

For the Total RNA Protocol, add_mix_pellet_aspirate has just two parameters: vol and source, the same ones as add_and_mix. (aspirate_supernatant is called with num_aspirations set to 3.)

For the DNA Extraction/Purification protocol, the function takes in seven parameters: 

def add_mix_pellet_aspirate(vol: float, source: list[protocol_api.Well], dest: protocol_api.Labware, num_aspirations: int, source_2: protocol_api.Labware, dest_2: protocol_api.Labware, remove_magnet=True):
    add_and_mix(vol, source, dest)
    new_loc = "5"
    if dest == rna_plate:
        new_loc = "6"
    protocol.move_labware(dest, new_location=protocol_api.OFF_DECK)
    protocol.move_labware(source_2, new_location=new_loc)
    protocol.delay(minutes=1)
    aspirate_supernatant(num_aspirations, source_2, dest_2)
    if remove_magnet:
        protocol.move_labware(source_2, new_location=protocol_api.OFF_DECK)
        protocol.move_labware(dest, new_location=new_loc)

From here each protocol just follows the steps given in their respective guides, making calls to the functions described as necessary. However, there are multiple points in the protocols that require steps to be performed manually by the user.

Every step that involves putting the sample plate on a magnetic separation device includes manual action. The protocol pauses so the user can put the magnetic block on the deck. Note that the robot sees the well plate and the well plate on the magnetic block as two separate pieces of labware, even though you're just putting the well plate on top of the magnet.

protocol.move_labware(sample_plate, new_location=protocol_api.OFF_DECK)
protocol.move_labware(magnetic_block, new_location="6")

The protocols will not continue until the user indicates in the Opentrons app that these manual steps have been completed.