Lidstrom:Ultracentrifuge

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== Basics ==  
== Basics ==  
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* [https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&_windowLabel=UCM_RENDERER&_urlType=render&wlpUCM_RENDERER_path=%2Fwsr%2Fsupport%2Ffrequently-asked-questions-faq%2Fcentrifugation%2Findex.htm FAQ page]
* If you desire 4<sup>o</sup>C during your spin cycle, you must allow the machine to cool to that point before it will allow you to press start.
* If you desire 4<sup>o</sup>C during your spin cycle, you must allow the machine to cool to that point before it will allow you to press start.
 +
=== Tubes collapsing===
* '''If you use too small of a volume (< half full) in the ultracentrifuge tubes, they will collapse!'''
* '''If you use too small of a volume (< half full) in the ultracentrifuge tubes, they will collapse!'''
** [[image:tubes collapsed in ultracentrifuge.jpg|thumb|center|Tubes with only 1/2 mL liquid collapse in ultracentrifuge.]]
** [[image:tubes collapsed in ultracentrifuge.jpg|thumb|center|Tubes with only 1/2 mL liquid collapse in ultracentrifuge.]]
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** It is hard to say how much is enough, but it seems to be about 1 mL.  1/2 mL '''definitely''' collapsed on [[User:Janet B. Matsen|Janet]] in 5/2013.  
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** 1/2 mL '''definitely''' collapsed on [[User:Janet B. Matsen|Janet]] in 5/2013 before I called tech support and learned the golden "1/2 full rule."
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** We should be able to find smaller-volume tubes for our rotor. ([https://www.beckmancoulter.com/wsrportal/wsr/research-and-discovery/products-and-services/centrifugation/optima-max-xp/index.htm link])
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** See below if you want to buy tubes for smaller volumes.
** You probably won't be able to get any sample out of a collapsed tube.   
** You probably won't be able to get any sample out of a collapsed tube.   
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** When they collapse it is very tricky to get them out.  Needle nose pliers are probably your best bet but it can still be very hard.
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** When they collapse it is very tricky to get them out.  '''Consult the rotor manual if you get tubes stuck.''' They recommend autoclaving the buckets upside down instead of trying to fish/pry the tubes out.
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* If the ultracentrifuge won't start and won't even try to start, open the door, lift the rotor up, place it back down, let it cool back down to your desired temp, and hold start.
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=== How balanced does it need to be? ===
=== How balanced does it need to be? ===
* Tech support 5/2013 [[User:Janet B. Matsen|JM]] said that you can balance oppositely placed buckets by eye.  There is no need to spend the time weighing down to the nearest milligram.  The centrifuge will stop itself if it is off balance.  The auto shut-off feature is conservative enough that you cannot hurt the rotor by having too imbalanced of samples.  You can, however, waste your time if you need to rebalance and restart the ultracentrifuge.
* Tech support 5/2013 [[User:Janet B. Matsen|JM]] said that you can balance oppositely placed buckets by eye.  There is no need to spend the time weighing down to the nearest milligram.  The centrifuge will stop itself if it is off balance.  The auto shut-off feature is conservative enough that you cannot hurt the rotor by having too imbalanced of samples.  You can, however, waste your time if you need to rebalance and restart the ultracentrifuge.
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=== The swinging buckets are numbered ===
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* You can match the bucket number to the corresponding position on the rotor, or do something else deliberately.
 +
** Janet's method: If my samples are ordered 1, 2, 3, 4 then I put them in bucket with their number. If the weights of 1 and 2 are balanced, and 3 and 4 are balanced I put the numbered buckets in a different position than their caps say.  It would be 1, 3, 2, 4 in the 1st, 2nd, 3rd, and 4th position.  I also make a sharpie label on each disposable tube to eliminate the possibility of mixing up samples in this process. 
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=== When it wont' try to start ===
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* If the ultracentrifuge won't start and won't even try to start, open the door, lift the rotor up, place it back down, let it cool back down to your desired temp, and hold start.
== Example Protocols ==
== Example Protocols ==
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== Parts & Info ==
== Parts & Info ==
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=== Tube options ===
* A fuller product spec sheet for the MLS-50: [[image:MLS-50 product sheet.jpg|center|MLS-50 product sheet]] from [https://www.beckmancoulter.com/wsrportal/bibliography?docname=BR-8101.pdf website PDF]
* A fuller product spec sheet for the MLS-50: [[image:MLS-50 product sheet.jpg|center|MLS-50 product sheet]] from [https://www.beckmancoulter.com/wsrportal/bibliography?docname=BR-8101.pdf website PDF]
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=== If you want to use different tubes ===
 
* Amanda has been buying 5mL Ultraclear tubes.  If you want to use < 2.5 mL, you need to buy smaller tubes.   
* Amanda has been buying 5mL Ultraclear tubes.  If you want to use < 2.5 mL, you need to buy smaller tubes.   
** You can't use conical unless you buy a support insert.
** You can't use conical unless you buy a support insert.
** Polycarbonate is more clear than polyallomer.
** Polycarbonate is more clear than polyallomer.
** You can't fill a thick walled tube < 1/2 full, even though they are thicker.  ([[User:Janet B. Matsen|Janet]] asked!)
** You can't fill a thick walled tube < 1/2 full, even though they are thicker.  ([[User:Janet B. Matsen|Janet]] asked!)
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[[image:UltraClear ultracentrifuge tubes.jpg|thumb|upright=1|UltraClear ultracentrifuge tubes]]
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* Janet is tempted by stainless steel tubes.  We should call tech support before buying.  Can you fill these < 1/2 full? [https://www.beckmancoulter.com/wsrportal/bibliography?docname=B2011-12564.pdf link]

Revision as of 09:40, 14 June 2013

Back to Protocols

Contents

Our System

Basics

  • FAQ page
  • If you desire 4oC during your spin cycle, you must allow the machine to cool to that point before it will allow you to press start.

Tubes collapsing

  • If you use too small of a volume (< half full) in the ultracentrifuge tubes, they will collapse!
    • Tubes with only 1/2 mL liquid collapse in ultracentrifuge.
      Tubes with only 1/2 mL liquid collapse in ultracentrifuge.
    • 1/2 mL definitely collapsed on Janet in 5/2013 before I called tech support and learned the golden "1/2 full rule."
    • See below if you want to buy tubes for smaller volumes.
    • You probably won't be able to get any sample out of a collapsed tube.
    • When they collapse it is very tricky to get them out. Consult the rotor manual if you get tubes stuck. They recommend autoclaving the buckets upside down instead of trying to fish/pry the tubes out.

How balanced does it need to be?

  • Tech support 5/2013 JM said that you can balance oppositely placed buckets by eye. There is no need to spend the time weighing down to the nearest milligram. The centrifuge will stop itself if it is off balance. The auto shut-off feature is conservative enough that you cannot hurt the rotor by having too imbalanced of samples. You can, however, waste your time if you need to rebalance and restart the ultracentrifuge.

The swinging buckets are numbered

  • You can match the bucket number to the corresponding position on the rotor, or do something else deliberately.
    • Janet's method: If my samples are ordered 1, 2, 3, 4 then I put them in bucket with their number. If the weights of 1 and 2 are balanced, and 3 and 4 are balanced I put the numbered buckets in a different position than their caps say. It would be 1, 3, 2, 4 in the 1st, 2nd, 3rd, and 4th position. I also make a sharpie label on each disposable tube to eliminate the possibility of mixing up samples in this process.

When it wont' try to start

  • If the ultracentrifuge won't start and won't even try to start, open the door, lift the rotor up, place it back down, let it cool back down to your desired temp, and hold start.

Example Protocols

  • To spin down the membranes and membrane-bound proteins in a crude extract, centrifuge for 45,000 RPM at 4oC for 1 hour. With our MLS-50 mushroom-shaped rotor, this is 163,000xg at raverage (108,000xg at rminimum).

Important Info about Rotor & Tubes

  • Tubes should never be less than half filled or they will collapse. - Tech support 5/2013 via Janet
    • This is true for every tube material and size.
  • You can wash and re-use tubes, however, deformed tubes will collapse.
    • Be careful to wash them with compatible liquids. See the manuals.
  • There are seals inside the swinging buckets (tube holders) that seal the sample when you screw the bucket lid tight. These are very important. The ultracentrifuge uses a strong vacuum to keep the samples chilled. If the seal fails, your sample will be sucked out of the swinging basket and end up in the vacuum. Then the centrifuge will stop because it gets off balance. There is no way to recover after this.
    • location = top of bucket (thing that sits on the mushroom)
    • Tech support said we should inspect monthly, replace them annually, and have spares on hand. I am skeptical, as this doesn't factor in how heavily the system is used. If one gets pinched/cracked then replace them all at the same time.
    • Item ordering number: 824412 O.

Parts & Info

Tube options

  • A fuller product spec sheet for the MLS-50:
    MLS-50 product sheet
    from website PDF
  • Amanda has been buying 5mL Ultraclear tubes. If you want to use < 2.5 mL, you need to buy smaller tubes.
    • You can't use conical unless you buy a support insert.
    • Polycarbonate is more clear than polyallomer.
    • You can't fill a thick walled tube < 1/2 full, even though they are thicker. (Janet asked!)
UltraClear ultracentrifuge tubes
UltraClear ultracentrifuge tubes
  • Janet is tempted by stainless steel tubes. We should call tech support before buying. Can you fill these < 1/2 full? link
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