Editing
Arichard:Protocols/size selection 150402
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!
==MDA amplicon size selection== ===Prep=== * For each sample pool: ** Place a 0.5 ml tube inside a low binding 1.5 ml tube. Use a 20 G needle to punch a hole in the bottom of the low binding 0.5 ml tube, then UV treat. ** UV 400 ul TE buffer. ** UV a nanosep tube. ** UV 1 ml 100% EtOH, 40 ul 3 M NaAc, and 1 ul GlycoBlue in a 1.5 ml tube. ** Chill 75% EtOH wash at -80 C before using. ===Protocol=== ====Size selection==== # Run pools up to 40 ul on 5 lane PAGE gels, 250 V for 20 minutes. Use 20 ul 6X Loading Dye (NEB), 2 ul Low Mass Ladder (Invitrogen). Use additional 0.5X TBE to equalize volumes between all pools. # Add 3 ul SYBR Gold to tray with 100 mL 0.5X TBE. Mix well before adding gel to avoid bright spots. # Incubate gel 2 minutes in tray on shaker. # Size select between 200 and 800 bp with scalpel. Load gel fragment into 0.5 ml tube (inside 1.5 ml tube). # Spin gel in tube-in-tube setup 2 minutes at 12,000 RPM. Remove and discard 0.5 ml tube. # Add 400 ul TE buffer and incubate shaking for 1 hour at 37 C. # Load into NanoSep tube and spin 3 minutes at 15,000 RPM. ====Ethanol precipitation==== # Load Nanosep flowthrough into EtOH precipitation mix and incubate at -80 C for 30 minutes. # Spin for 30 minutes at 10,000 RPM at 4 C. Remove and discard supernatant. # Wash with 750 ul cold 75% EtOH and spin 5 minutes at 10,000 RPM at 4 C. # Remove wash and let dry 5-10 minutes. Resuspend in 20 ul H2O. Store at -20 C until sequencing.
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