Editing
Noi/NOTES/2014-12-17
(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!
=== 6) Bisulfite conversion === * I performed bisulfite conversion using MethylCode™ Bisulfite Conversion Kitfrom LifeTechnologies and used the same procedure following manufacturer's instruction and elute with 32ul elution buffer. '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Prepare 4 tubes of CT Conversion Reagent, by adding 850ul H2O, 50ul of Resuspension Buffer, and 300ul of Dilution Buffer to CT Conversion Reagent (for 25ul DNA sample --> reduce H2O from 900 to 850) :- Add125ul of complete CT Conversion Reagent to adaptor ligated DNA (no sample transfer to the new tube) :- Mix by pipetting 10X with multi-channel pipette :- Spin down the plate at 2,000rpm for 1min :- Incubate following below program ::- 98°C for 10 minutes (DNA denaturation) ::- 64°C for 2.5 hours (Bisulfite conversion) ::- 4°C storage for up to 20 hours or continue to desulfonation '''''<span style="color:crimson"><u>Prep</u></span>'''''<br> - Mix 600:1 ratio of Binding Buffer and 10ng/ul tRNA<br> - For 33.5 rxn, I mixed 20.10mL of Binding Buffer with 33.5 ul of 10ng/ul tRNA : - Add 601ul of Binding Buffer/tRNA mix to the column :- Bind DNA to column by transfer bisulfite-treated DNA to the column and mixing by pipetting up and down for 5X. :- Spin down 14,000rpm for 30sec. <u>Discard spnt</u> :- Wash with 100ul Wash buffer :- Spin down 14,000rpm for 30sec :- ''Incubate with 200ul of Desulphonation Buffer for 15min'' :- Spin down 14,000rpm for 30sec :- Wash column with 200ul Wash Buffer. :- Spin down 14,000rpm for 30sec. <u>Discard spnt</u> :- Wash the column with 200ul Wash Buffer. :- Spin down 14,000rpm for 2min :- Elute converted DNA with warm (~60C) 32ul Elution Buffer. Incubate column with Elution Buffer at room temp for 2min. :- Spin down at 14,000rmp for 1min. This should have ~30ul DNA left for PCR
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