http://2009.igem.org/wiki/index.php?title=Team:Washington/Notebook/2mL_purification&feed=atom&action=historyTeam:Washington/Notebook/2mL purification - Revision history2024-03-29T15:47:18ZRevision history for this page on the wikiMediaWiki 1.16.5http://2009.igem.org/wiki/index.php?title=Team:Washington/Notebook/2mL_purification&diff=100030&oldid=prevDunbar at 05:12, 15 October 20092009-10-15T05:12:21Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Spin column for 5min at 500rpm </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Spin column for 5min at 500rpm </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#FLOW THROUGH IS YOUR PURIFIED PROTEIN</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#FLOW THROUGH IS YOUR PURIFIED PROTEIN</div></td></tr>
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</table>Dunbarhttp://2009.igem.org/wiki/index.php?title=Team:Washington/Notebook/2mL_purification&diff=100016&oldid=prevDunbar at 05:09, 15 October 20092009-10-15T05:09:25Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Supernatant Protein Purification, 2mL ====</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==== Supernatant Protein Purification, 2mL ====</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Inoculate 50mL culture of TB with ~750uL overnight culture</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>#Inoculate 50mL culture of TB with ~750uL overnight culture</div></td></tr>
</table>Dunbarhttp://2009.igem.org/wiki/index.php?title=Team:Washington/Notebook/2mL_purification&diff=99934&oldid=prevDunbar: New page: ==== Supernatant Protein Purification, 2mL ==== #Inoculate 50mL culture of TB with ~750uL overnight culture #Grow a 37c until OD600: 0.4 #Inoculate cells with IPTG so that the final concen...2009-10-15T04:10:55Z<p>New page: ==== Supernatant Protein Purification, 2mL ==== #Inoculate 50mL culture of TB with ~750uL overnight culture #Grow a 37c until OD600: 0.4 #Inoculate cells with IPTG so that the final concen...</p>
<p><b>New page</b></p><div>==== Supernatant Protein Purification, 2mL ====<br />
#Inoculate 50mL culture of TB with ~750uL overnight culture<br />
#Grow a 37c until OD600: 0.4<br />
#Inoculate cells with IPTG so that the final concentration is 0.5mM (25uL of 1M IPTG for 50mL culture)<br />
#Grow cultures until OD600: 4, or use time points if looking for comparison in protein in supernatant<br />
#Prep NiNTA columns <br />
##Place micro-centrifuge columns in collection tubes<br />
##In micro-centrifuge columns add 200uL NiNTA beads<br />
##Add 500uL PBS, aspirate to thoroughly rinse columns <br />
##Spin columns with collection tubes for 30sec at 500rpm<br />
#Transfer 2mL of growing culture to eppendorf tube<br />
##Spin tube for 20min at 8000 rpm<br />
##Remove supernatant, carefully as to not disrupt the pellet and set aside<br />
#Bind Protein to column<br />
##Add 500uL supernatant to the column<br />
##Spin for 30sec at 500rpm<br />
##Discard flow through<br />
##Repeat 1-3 until all supernatant has run though the column<br />
#Wash column<br />
##Apply 500uL PBS to column, aspirate to suspend beads<br />
##Spin column for 30sec at 500rpm, discard flow through<br />
##Repeat 1-2<br />
#Elute protein off of column<br />
##Make PBS with 1mg/mL BSA and 100uM imidazole<br />
##Add 200uL PBS + 1mg/mL BSA + 100uM imidazole to column aspirate to mix beads<br />
##Let sit for 2 min<br />
#Place Column in clean collection tube<br />
#Spin column for 5min at 500rpm <br />
#FLOW THROUGH IS YOUR PURIFIED PROTEIN</div>Dunbar