Daniel:Protocols/AmiconUltra: Difference between revisions

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Amicon Ultra Centrifugal filters are microcentrifuge filters that allow you to purify and buffer exchange various MW cutoff biomacromolecules. They use a MW cutoff filter, say 10 kDa, to allow dialysate through while not passing the biomacromolecules. They can be used for buffer exchange via the following general protocol:
Amicon Ultra Centrifugal filters are microcentrifuge filters that allow you to purify and buffer exchange various MW cutoff biomacromolecules. They use a MW cutoff filter, say 10 kDa, to allow dialysate through while not passing the biomacromolecules. They can be used for buffer exchange via the following general protocol:


[[Image:CentrifugalFilters.png|400px]]
<gallery perrow=2 heights=300px widths=400px>
File:CentrifugalFilters-Purification.png|Purification/Concentration
File:CentrifugalFilters.png|Buffer Exchange
</gallery>


==Protocol-Filtration==
==Protocol-Filtration==
Line 22: Line 25:


<ol>
<ol>
<li><li>Add X uL sample and new buffer to 500 uL total volume</li>
<li>Add X uL sample and new buffer to 500 uL total volume</li>
<li>Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)</li>
<li>Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)</li>
<li>Discard flow through and add 500 uL new buffer</li>
<li>Discard flow through and add 500 uL new buffer</li>
<li>Repeat steps 2 and 3 as many times as desired</li></ol>
<li>Repeat steps 2 and 3 as many times as desired</li></ol>
===Dialysis Table===
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#FFCC66;font-size:12pt;font-weight:bold" align="center"
| width="120" height="30" | Starting Sample Volume
| width="110" | 500 uL dilution
| width="110" | Original Buffer Concentration
| width="110" | Centrifuge Time
|- style="font-size:12pt" align="center"
| align="center" height="15" | 50
| align="center" | 10
| align="center" | 10%
| 15 min
|- style="background-color:#BFBFBF;font-size:12pt" align="center"
| align="center" height="15" | 20
| align="center" | 25
| align="center" | 0.4%
| 15 min
|- style="font-size:12pt" align="center"
| align="center" height="15" | 20
| align="center" | 25
| align="center" | 0.016%
| align="center" | 15 min
|}
==Spin Times and Concentration==
[[Image:AmiconSpinTimeTable.png|800px]]

Latest revision as of 21:52, 13 May 2017

Amicon Ultra Centrifugal Filters[edit]

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Amicon Order Page/Information

Amicon Ultra Centrifugal filters are microcentrifuge filters that allow you to purify and buffer exchange various MW cutoff biomacromolecules. They use a MW cutoff filter, say 10 kDa, to allow dialysate through while not passing the biomacromolecules. They can be used for buffer exchange via the following general protocol:

Protocol-Filtration[edit]

  1. Add X uL sample and buffer to 500 uL total volume
  2. Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)
  3. Discard flow through tube and get a new one
  4. Invert filter inside flow through tube and spin down at 14000xg for 1 minute
  5. Alternately, extract sample using pipette

Protocol-Dialysis[edit]

  1. Add X uL sample and new buffer to 500 uL total volume
  2. Spin down at 14000 x g for appropriate time (15 minutes for 22 uL volume)
  3. Discard flow through and add 500 uL new buffer
  4. Repeat steps 2 and 3 as many times as desired

Dialysis Table[edit]

Starting Sample Volume 500 uL dilution Original Buffer Concentration Centrifuge Time
50 10 10% 15 min
20 25 0.4% 15 min
20 25 0.016% 15 min

Spin Times and Concentration[edit]

File:AmiconSpinTimeTable.png