Editing
Daniel:Notebook/ComboLock/2016-5-26
(section)
Jump to navigation
Jump to search
Warning:
You are not logged in. Your IP address will be publicly visible if you make any edits. If you
log in
or
create an account
, your edits will be attributed to your username, along with other benefits.
Anti-spam check. Do
not
fill this in!
==Protocol== <ol> <li>Prep</li> <ol type="A"> <li>Resuspend VIM-PA in 588 uL nfH2O</li> <li>Resuspend VIM-PB in 532 uL nfH2O</li> </ol> <li>Antibody Activation</li> <ol type="A"> <li>Resuspend VIM with 100 uL PBS (final conc 1 mg/mL)</li> <li>Do this twice:</li> <ol type="a"> <li>Equilibrate a 40 kDa spin column 4 times with 100 uL PBS, (100 mM pH 7.3) <ol type="i"><li>Spin at 1000xg for 2 min</li></ol> <li>Add 20 uL antibody to the column and spin for 3 min at 1000xg; collect in a new tube</li> <li>Add 2 uL sulfo-SMCC to sample</li> <li>Incubate for 2 hr at 4C with 3 times intermittent mixing</li> <li>Equilibrate a new 40 kDa column with 100 mM phosphate buffer with 20 mM EDTA</li> <li>Transfer antibodies to new column and spin for 3 min at 1000xg</li> </ol> </ol> <li>Oligonucleotide Activation</li> <ol type="A"> <li>For VIM-PA and VIM-PB:</li> <ol type="a"> <li>Add 13 uL 100 uM oligonucleotide to tube</li> <li>Add 22 uL 40 mM DTT</li> <li>Incubate at 95C for 2 min followed by 1 hr at 37C</li> <li>Add 165 uL PBS with 20 mM EDTA</li> <li>Remove excess DTT using two consecutive [[Media:Zeba_Spin_Desalt_Colomn_7K_MWCO_UG_Manual.pdf|Zeba 7 kDa]] columns equilibrated with 100 mM PBS (10X PBS)</li> <ol type="a"><li>Spin at 1500xg for 1 minute for equilibrium; 2 min for sample</li></ol> </ol> </ol> <li>Ab-Oligo Mixing</li> <ol type="A"><li>For VIM-PA and VIM-PB:</li> <ol type="a"> <li>Mix SMCC-treated antibodies (22 uL) with DTT-treated oligonucleotides (35 uL)</li> <ol type="i"><li>Amounts to 10X molar excess of oligo to Ab</li> <li>Transfer to pre-wet [[Media:SlideALyzer_MINI_Dialy_Unit_UG_Manual.pdf|Slide-A-Lyzer Mini 7 MWCO dialysis cup]]</li> <li>Dialyze in 1 L PBS with 5 mM EDTA at 4C for 2 days with one buffer exchange to PBS</li> <li>Dilute probes to 75 ug/mL in PBS buffer with 4 mM EDTA, 35 ug/mL ssDNA, 0.1% fish gelatin, and 20 mM Tris HCl</li> </ol>
Summary:
Please note that all contributions to ZhangLabWiki may be edited, altered, or removed by other contributors. If you do not want your writing to be edited mercilessly, then do not submit it here.
You are also promising us that you wrote this yourself, or copied it from a public domain or similar free resource (see
ZhangLabWiki:Copyrights
for details).
Do not submit copyrighted work without permission!
Cancel
Editing help
(opens in new window)
Navigation menu
Personal tools
Not logged in
Talk
Contributions
Create account
Log in
Namespaces
Page
Discussion
English
Views
Read
Edit
View history
More
Search
Navigation
Main Page
Current events
Recent changes
Random page
Investigators
Matt Cai
Song Chen
Eric Chu
Dinh Diep
Elizabeth Duong
Shicheng Guo
Alan Fung
Daniel Jacobsen
Blue Lake
Huy Lam
Alice Li
Andrew Richards
Brandon Sos
Chris Wei
Yan Wu
Kun Zhang
Tools
What links here
Related changes
Special pages
Page information