Running a protocol - Standard user - 6.4 - Preparing firefly

The first screen reminds you to empty firefly of any labware from a previous protocol. Click 'Next' when it is empty.

The screen will show your firefly's configuration; the number of dispense heads will be correct and the shaker and thermal modules will be greyed-out on Non-genomics instruments.

If your protocol has an initial temperature setpoint for either the plate or the reservoir thermal
blocks, you will start heating or cooling them as part of set up. Use 'Set temperature' to begin
heating or cooling. 'Next' is not enabled until the block reaches the set temperature.

If the protocol requires the dispense head and it already contains syringes from a previous
protocol, the next screen will prompt you to remove them. You do not have to do this e.g., if you
are running the same protocol again and your lab procedures do not require you to change the
syringes.

6.4.1 Loading firefly

The next screen starts the instrument set up process, by showing the completed deck set-up.
firefly will guide you through loading each item in the following screens. Each step shows one
loading operation and has options for 'Skip', Get Access', 'Back' and 'Next'. When the buttons are
greyed out, firefly is operating and not ready for user access.

You can select 'Skip' to move through some or all of the sections of the loading sequence more
quickly if you are an experienced firefly user and you have permission to do so.  

 

Ensure you have all your items ready e.g., cold plates or reservoir inserts are chilled, tip sets or
strips are loaded into tip cassettes and syringes are assembled, then click 'Next'.

Follow the on-screen instructions for loading each item and the instructions below for physical
loading.

1. Select 'Get Access', unless you are loading syringes in which case follow the specific
   instructions below. Wait for firefly heads and decks to move to safe positions before you
   open the door.
2.  You may need to scan barcodes on reagents, tip sets, plates, syringes or reservoirs.
   
   
3. Open the door.
4. Load your materials (see specific instructions below).
   IMPORTANT: Be sure to put each item on the deck location specified. firefly cannot validate that you have done this correctly so you must follow the instructions exactly or your protocol will fail.
5. Close the door.
6. Click 'Next' on the wizard, or 'Back' if you are loading materials in a different order to that
   suggested and repeat the loading process for the next item.

When all materials and consumables are loaded, you will see 'Setup complete'

6.4.2 Loading tips

To load a full array of 96 or 384 tips:

1. Open the tip cassette.
2. Slide the tips into the tip cassette. For 96 tips, the B1 tip must be at the open end of the hinge for the tips to fit securely in the cassette. As the tips are being positioned into the cassette, lift the collar holding the tips together just enough to clear the grilles of the cassette.
   
   Avoid dropping the tips into the closed cassette from above the cassette, as this action may cause the tips to touch and contaminate the cassette and/or tips.
   
   
3. Close the gate. Don't tip the cassette upside down - the tips will fall out.
4. Place the tip cassette into the tip stand.

IMPORTANT: The tip stand must be clean and free of dust before tips are loaded into it. Failure to ensure this may result in dust and debris being transferred to the tips.

 5. Scan the barcode, if required.

6. Place the tip set on the lower deck in the position shown on the Load Tips screen.

The word "FRONT" is present on each cassette and it must be facing you when the tip set is
loaded onto the deck. 

To load one or more tip strips (all sizes): 

1. Select the correct strip tip adaptor (16 or 8) for your plate and tips. For 96 format tips, use the
8 adaptor below.

For 384 format tips, use the 16 adaptor below.

Place the strip tips adaptor on the tip cassette, to keep the strip tips in position.

2. Slide the tip strip into the tip adaptor, so that it is at the far right position (column 12 for 96
wells).

3. If required, add additional strip tips, working from the right. Close the tip cassette gate. Don't tip the cassette upside down - the tips will fall out.

4. Place the tip cassette into the tip stand.

IMPORTANT: The tip stand must be clean and free of dust before tips are loaded into it. Failure toensure this may result in dust and debris being transferred to the tips.

5. Place the tip set on the lower deck in the position shown on the Load Tips screen.

The word "FRONT" is present on each cassette and it must be facing you when the tip set is loaded onto the deck.

6.4.3 Loading plates, risers, lids, thermal blocks and magnetic blocks

1. Scan the plate barcode, if required.

2. Load plates, risers, lids, magnetic blocks or thermal blocks in the upper or lower deck positions specified in firefly setup. The deck locations are labelled.

For plates, A1 is positioned at the back left corner: this is labelled on each deck position.

Warning: if you are loading plates containing reagents, you may wish to use lids or non-pierced seals while loading but you must:

• Remove non-pierced seals before starting protocol execution.
• Manually remove lids, or include a Move or Take Off step in the protocol to remove the lids.

3. Ensure your plates are seated firmly in position and flat. There is a plate clamp at the bottom
right corner which engages after loading to keep the plate stationary but for it to operate
correctly, the plate must be positioned correctly on the deck. 

6.4.4 Removing syringes

If firefly has previously run a protocol, it may have syringes loaded from that protocol. firefly
software detects this and prompts you to remove them.

You will need an empty reservoir for each syringe, to purge them of any remaining liquid before
removing the syringes.

However, if you are running the same protocol repeatedly, you may not need to replace the
syringes to avoid contamination, in which case click 'Next' and continue, reusing the same
syringes.

To remove syringes:

1. Use 'Get access' to safely access the dispense head, and open the doors. Load empty
reservoirs for waste liquid.

2. Click the Remove button to dispense all liquid in the syringe and home the piston rods to allow safe removal of the syringe. 
3. firefly shows a final popup for you to confirm that the reservoirs are ready for use in purging.

firefly then purges the syringes, ready for removal.

IMPORTANT: don't try and remove the syringe without using the Remove function first; if the piston rod is still engaged, you will damage your instrument.

4. Remove syringes by rotating them clockwise, which will free them from the track. Remove the
liquid waste. When you have removed all the syringes, the 'Remove' function will no longer be
available (greyed out button).

5. Select 'Next' to continue.

6.4.5 Assembling syringes

Before you can load syringes, you will need to assemble them:

1. Place one plunger in the syringe, point down, as shown above.
   CAUTION: Make sure you only place one plunger in the syringe. If you insert two, you
   will break firefly, and need a service engineer visit to get it working again.
2. Gently press it far enough into the syringe that it is completely enclosed. It doesn't matter
   whether it's straight, or how deep in the syringe it is when you load it.

6.4.6 Loading syringes

Before you load a syringe, you may need to scan its barcode.

 The Load Syringes screen directs you to load each syringe in turn.

1. Load syringes into the positions highlighted on the setup page. The dispense head has three tracks. 3 head instruments have one syringe in each track, 6 head instruments have two. Position S1 is the syringe furthest back and furthest left, as you look at the dispense head,
circled in the illustration below.

2. Slide each syringe into the correct track.

3. For a back row syringe, slide the syringe until it stops, then push upwards and rotate 90
degrees counter-clockwise.

For a front row syringe, push upwards while sliding the syringe into the track; when it pushes
into a hole, then rotate counter-clockwise.

6.4.7 - Loading reagent reservoirs

The initial Load Reservoirs screen is an overview of all reservoirs required. Only R1 - R3 are used for a 3 head instrument.

Ensure you have the correct tray for your reservoirs. If your protocol requires high volume reservoirs, you will need to use the correct tray, and the correct inserts for any standard or LDV
reservoirs.

After the overview screen, you will see the instructions for each reagent and reservoir in turn.

Scan the reagent and reservoir barcodes as directed, if they are used.

The detailed instructions specify the liquid to be loaded in each reservoir. and the volume required.

To load the reservoir loading tray:

1. Remove the reservoir loading tray.
2. Place either a high volume, a standard or a 1.5mL Low Dead Volume (LDV) reservoir in each position as specified in the loading screen. Use 'Next' to see the details of all reservoirs to be loaded.
3. Fill each reservoir according to the instructions in the wizard. Any inaccuracy in the volume
   that is loaded into the reservoirs could cause a problem with the dispense. To avoid issues,
   use a calibrated pipette and good pipetting technique when loading reservoirs with liquid.
   The volume includes the calculated dead volumes in the reservoir and the syringe, in addition to the volumes dispensed. 10mL reservoirs have molded 5mL and 10mL (the maximum fill) level marks as a guide.
4. If you are using high volume reservoirs, you may also be using lids to lessen evaporation. Make sure you have put the lids on the correct way round, with the hole in the lid facing the
   center of the tray, as shown below.
   
   IMPORTANT: firefly syringes will crash into the reservoir lids if the holes are not correctly aligned with the tip positions.
5. Carefully move the reservoir tray, with the filled reservoirs, back to firefly.
6. Use the guide slots to reposition it in the instrument. You should hear a click when the tray
   reaches its correct position. 

Next Article - Running a protocol - Standard user - 6.5 - Running your protocol