(Last updated: April 2020)

Using the PlateLoc (automated plate sealer)

The PlateLoc is a part of the Bravo set up, but it can be separately used to seal skirted 96-well and 384-well plates.

1. Depending on the type of plate being sealed, there is a different removable platform necessary for a tight seal.

• 290: U-bottom plates, or any plate with some distance between the bottom of the wells and the edge of the skirt.
• 180: PCR plates, or any plate where the bottom of the wells line up with the edge of the skirt.

2. Place the plate on the deck with the correct platform. Make sure the plate is well-placed and fits on the deck snugly.

3. Press the “Run” button of the right side of the PlateLoc screen. The pressurizer under the bench may kick in (and make a racket).

4. The plate will be hot after being sealed, so take care to not burn yourself when removing it from the deck.

Note that because this is a heat-based processed, not all plate types can be used with the system (some plate types would melt).

Making more diluted PCR plates

• If the diluted PCR plates (labeled “rePCR”) in the racks on Freezer #1 have wells that are running low or completely empty, check the other large DRSC -20 freezer for the PCR plate with the same number (labeled “PCR”).
• These plates are the original PCR plates with concentrated DNA. Dilute the PCR plates 1:100 to make the rePCR plates.
  • Make sure to use nuclease-free water.
  • Make 200uL of each reagent. In this case, add 2uL PCR product to 198uL nuclease-free water per well.

General lab notes

• Keep track of PCR and IVT kits inventory, as well as how many boxes and sleeves of each assay-ready plates are currently in the lab. Assay-ready plates are generally in boxes on the top shelves of Bay 3.
• If working on a larger project, keep track of the number of robot tip boxes for the MultiProbe and the Bravo. Large projects go through tips very quickly.

General gel notes

• The smaller tray (fits two 20-well combs) can hold 200mL of agarose to make a thick gel.
• The larger tray (fits four 50-well combs) can hold 400mL of agarose to make a thick gel.
• We reuse the Erlenmeyer flasks by the trays and rigs in Bay 3 to make and run gels. Each flask has a stir bar inside for the stir plate by the microwave.
• 2x TBE is in the back of the Perrimon Lab above the sink. Agarose, weigh boats, and scales are in a little nook in the Perrimon lab.
• Ethidium bromide is located in a labeled box next to the microwave. Add 4uL ethidium bromide per 200mL of 1% agarose gel.
• Cut off any extra gels and save for later use by wrapping it in plastic wrap (which can be found in the drawer right by the large biohazard bin next to the MultiProbe) and putting it in the mini 4 degree fridge under the bench with the rigs.
  • Be sure to use the gel within the week, or throw it out. Otherwise it dries into jerky.

Making a gel

For the IVT pipeline, roughly 1% gels are used. See the table below for gel recipes.

1. Add TBE and water to the Erlenmeyer flask, followed by the agarose. Swirl the flask to mix the contents.

2. Microwave the flask until the agarose has completely dissolved. 3 minutes, stir, then 1 more minute is usually sufficient. Remove the stir bar before microwaving.

3. Add the stir bar and stir the gel for ~15-20 min on the stir plate by the microwave until the glass isn’t hot, but still warm.

4. Add the ethidium bromide. While the gel is still stirring, lower the pipettor into the flask to ensure the ethidium bromide is added directly to the gel. The EtBr should be mixed in within a few seconds. Dispose of the pipette tip in the special EtBr waste container on top of the microwave.

5. Slowly pour the gel into the prepared tray (combs and stoppers added, ends taped up). Leave the gel to cool and solidify.

Quartzy Notes

Quartzy is the online system we use in the DRSC/TRiP (and in the Perrimon Lab) to order common lab supplies. The system keeps track of past orders, so it is easy to re-order the same supplies by looking through past inventory on Quartzy. The lab administrator will approve the order within a couple days; you can track the status of your orders on Quartzy as well.

Some common items (like Eppendorf tubes, the PCR kit used in the DRSC, and Qiagen kits) are not ordered through Quartzy but downstairs through the Biopolymers facility. Ask Stephanie for the login information for their website.

To place an order:

1. Check if the item has been ordered in the past. Under the “Requests” tab on the top left-hand side, click the “Received” sub-tab. Enter keywords of the item into the “Search received requests” bar on the left sidebar.

2. If the item is not in the “Received” sub-tab, click the “Inventory” tab next to “Requests.” Enter keywords into the “Search inventory” bar on the left sidebar.

3. Once you find your item, hit the blue “Request” button on the right. Confirm the request on the next screen.

• We only place orders through Quartzy with our vendors of choice; we do not swap vendors to Quartzy, even if the site prompts you to do so because an item is presumably cheaper. The lab has its own discounts in place with the external vendors independently of Quartzy.

4. If the item is not in Quartzy and has never been ordered before, create a new request in Quartzy.

• First look up the item on the website of your external vendor of choice. Companies we frequently order consumables from include VWR, ThermoFisher, and New England Biolabs. Note the exact name of the item, the catalog number, and the URL.
• On Quartzy, go to the “Requests” tab and hit “Add Request” on the left sidebar. Enter in the prompted information. If the correct item pops up, hit “Request.” If not, enter in the relevant info on the next form. Make sure to order directly from the vendor, not the Quartzy shop.

5. After receiving the order, go back into Quartzy and mark the order “Received.” You can find all placed orders pending arrival in the “Ordered” sub-tab under “Requests.”