Smolke:Protocols/Guidelines: Difference between revisions
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If machine is off: | If machine is off: | ||
*Turn on machine by touching | *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). | ||
Line 14: | Line 14: | ||
*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== | ||
Line 21: | Line 34: | ||
==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: | ||
# 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. |
Latest revision as of 14:48, 18 November 2012
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:
- 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.
- 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.