Smolke:Protocols/Guidelines

Start Up

Instructions for machine start up and various other functions are on the lamenated guides near the VYB.

If machine is off:

• Turn on machine by touching the screen.
• Logon and put the machine into data acquisition mode.
• Run back flush (~16 min).
• Run clean with 1% hypochlorite (~10 min).
  • NOTE: 100% bleach = 15% hypochlorite.
  • NOTE: Clean only runs if machine is in the single tube mode.
• Run calibration beads.

If machine is already on, assume it is not clean:

• Run back flush (~16 min).
• Run clean with 1% hypochlorite (~10 min).

Calibration

Calibrate the machine using the MACS Quant beads when the machine asks (notification at the bottom of the screen, near the left).

• Put the machine in single tube holder mode.
• Briefly vortex the beads to mix. Beads are in the fridge under the Biacore, on the bottom shelf.
• Click the barcode icon near the top screen.
• Scan the barcode on the bead bottle.
• Follow the instructions on the screen.
• If calibration does not pass:
  • Make sure the lasers have had >30 min to warm up. If this is not the issue:
  • Warm up 1% hypochlorite to 37oC, re-run with warm bleach, and re-run beads. If this does not work:
    • Contact VYB support under 'Tools' tab > 'MACSQuant live support'.
    • Let Kathy know ASAP.

Shut Down

• When finished, ALWAYS make sure there is ENOUGH BUFFER and ENOUGH SPACE in the waste container. Otherwise, shut down will stall and leave sticky, dead, clog-inducing samples in the machine until someone notices.
• Shut down the machine unless another user is signed up within a couple of hours after you finish, but check buffer and waste in case later user cancels.
• Machine will automatically attempt to shut down ~2 hrs after its last fluidics function. However, you must still check buffer and waste before leaving the machine.

96-Well Plates

• The VYB can only run ONE full 96-well plate if:

1) It has a FULL bottle of running buffer and an EMPTY waste bottle. 2) Flow rate is set to HIGH. 3) Mode is set to SCREEN. 4) Sample volume run is set to <50 uL.

• The cooler for 96-plate is kept at the bottom shelf of deli fridge. -YHW
• Make sure the running buffer bottle is full and waste bottle is empty before EACH 96-well plate.
• NOTE: In the future, there will be larger buffer bottles & this may no longer be a problem.

Empty Buffer Bottles

• Rinse with water.
• Remove the label and lid.
• Recycle (blue atrium bin).

Filtering Samples

ALWAYS FILTER YOUR SAMPLES.

• Nylon filter sheets (http://www.amazon.com/Microns-Square-Opening-Thread-Diameter/dp/B000FN0OG2/ref=pd_sbs_indust_5)
  • Available in zip-lock bag above VYB bench.
  • Clean-up:
    • Mammalian samples: Soak in 10% bleach for about 1 day, then soak in water about 1 day, then dry and return sheets to bag.
    • Yeast samples: Soak in water for a couple of days, then dry and return sheets to bag. (YHW: I usually spread ethanol on it while drying)
• Cell strainers (BD Falcon Cat. No.: 352340)
  • Not consistently stocked. Order if needed.
  • Disposable.

Quirks

• The software is frozen.
  • If you are running a sample, leave the machine alone. It is probably busy recording a lot of data and will recover once it finishes (up to ~15 min).
• I can't run 'Clean'.
  • Make sure the machine is set in the single tube mode.
• My samples look weird in FlowJo.
  • In FlowJo, adjust the file properties to match the decade settings used on the VYB.