Smolke:Protocols/Guidelines

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(Filtering Samples)
Current revision (17:48, 18 November 2012) (view source)
(96-Well Plates)
 
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If machine is off:
If machine is off:
-
*Turn on machine by touching to screen.
+
*Turn on machine by touching the screen.
*Logon and put the machine into data acquisition mode.
*Logon and put the machine into data acquisition mode.
*Run back flush (~16 min).
*Run back flush (~16 min).
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*Run back flush (~16 min).
*Run back flush (~16 min).
*Run clean with 1% hypochlorite (~10 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==
==Shut Down==
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==96-Well Plates==
==96-Well Plates==
-
*The VYB can only run ONE full 96-well plate if it has a FULL bottle of running buffer and an EMPTY waste bottle.
+
*The VYB can only run ONE full 96-well plate if:
 +
# It has a FULL bottle of running buffer and an EMPTY waste bottle.
 +
# Flow rate is set to HIGH.
 +
# Mode is set to SCREEN.
 +
# 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.
*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.
*NOTE: In the future, there will be larger buffer bottles & this may no longer be a problem.
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**Clean-up:  
**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.
***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 EtOH on it while drying)
+
***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)
*Cell strainers (BD Falcon Cat. No.: 352340)
**Not consistently stocked. Order if needed.
**Not consistently stocked. Order if needed.

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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