Smolke:Protocols/Guidelines

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(96-Well Plates)
Current revision (16:48, 18 November 2012) (view source)
(96-Well Plates)
 
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==96-Well Plates==
==96-Well Plates==
*The VYB can only run ONE full 96-well plate if:
*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.
+
# It has a FULL bottle of running buffer and an EMPTY waste bottle.
-
2) Flow rate is set to HIGH.
+
# Flow rate is set to HIGH.
-
3) Mode is set to SCREEN.
+
# Mode is set to SCREEN.
-
4) Sample volume run is set to <50 uL.
+
# Sample volume run is set to <50 uL.
*The cooler for 96-plate is kept at the bottom shelf of deli fridge. -YHW
*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.
*Make sure the running buffer bottle is full and waste bottle is empty before EACH 96-well plate.

Current revision

Contents

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.
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