Editing
Athurva Gore/LabNotes/2011-4-8
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!
=Bisulfite Biotin-Binding= * Ran gel for Bisulfite-converted bead-bound DNA and normal bead-bound DNA: [[File:04072011_Low_Input_Bisulfite_FAILED.jpg|500px]] * Bisulfite-converted libraries did not work. ** DNA was definitely attached to beads, as normal amplification worked. ** DNA must have been detached ===Can we modify the experiment to still allow attachment of beads?=== * According to Invitrogen website [http://www.invitrogen.com/site/us/en/home/brands/Dynal/Streptavidin-Coupled-Dynabeads/dynabeads_streptavidin.html#How%20do%20I%20dissociate%20my%20biotinylated%20molecule], bead-detachment can occur when: ** Heat in formamide or basic solution at 95 for 2 min or 65 for 5 min ** Boil beads ** Basic solutions, formamide, EDTA, and heat in general are bad ** Specific detachment percentages: [http://www.invitrogen.com/site/us/en/home/brands/Dynal/Streptavidin-Coupled-Dynabeads/elution_of_the_streptavidin.html] * '''What could be causing detachment?''' ** Bisulfite conversion involves adding an acidic solution with heat, incubation for a very long time ** Desulphonation buffer is a strong base; could be detaching DNA ** Other problems? * '''Possible solutions:''' ** We can perform bisulfite conversion of the DNA first, and then attempt to bind beads *** Potential pitfall: Bisulfite might not be a good buffer for binding, could inhibit bead-capture *** Could we eliminate this problem by either ****splitting each bisulfite reaction into multiple bead-binding reactions or ****using less CT-conversion reagent (instead of 130 uL, use a lot less since we have much less DNA?) *** One other commercial protocol uses IPure beads to capture bisulfite-converted DNA [http://www.diagenode.com/media/catalog/file/MA_MBI-V1_11_05_10.pdf] *** They just use a longer binding time; we can bind for 30 min to an hour to try *** '''DO THIS TOMORROW''' ** We can skip desulfonation step according to a published paper: [http://nar.oxfordjournals.org/content/35/1/e4.full?searchid=1&HITS=10&hits=10&resourcetype=HWCIT&maxtoshow=&RESULTFORMAT=&FIRSTINDEX=0&fulltext=tetzner] *** Can use 30 min at 95 C as initial step in PCR; desulphonates DNA successfully without use of a base *** Could be a big problem; heat could greatly damage DNA if exposed for that long
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