Editing
Sam:LabNotes/Microbiome-new/2009-5-22
(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!
==Procedures== *Hybridization **Prepare four wraps for 1.5-mL tubes **The fixed cells (10^8) were washed with 0.5 mL PBS(at RT) twice and resuspended in 200 uL of hybridization buffer. Hybridize at 46C for 1.5 hr without shaking. (Use eppendorf isothermo mixer but don't turn on the mixing function). H-buffer(0.9 M NaCl, 20 mM Tris-HCl, 0.01% SDS) H2O 88 uL 2M NaCl 90 uL 2M Tris 2 uL 0.1% SDS 20 uL --------------- 200 uL *Washing **Pellet the labeled bacteria by 8000 x g, 3 min. During this time, prewarm the washing buffer at 48C. **Remove the supernatant. Resuspended in 500 uL washing buffer and incubate at 48C for 20min without shaking. '''NOTE: The pellet is difficult to be resuspended into washing buffer. Pipetting the pellet carefully and repeatedly for ~20 times using p1000 tips. W-buffer (180 mM NaCl, 20 mM Tris, 0.01% SDS, 5 mM EDTA) H2O 395 x2 2M NaCl 45 2M Tris 5 0.1% SDS 50 0.5M EDTA 5 ------------------- 500 uL *PBS washing **Pellet the washed bacteria by 8000 x g, 3 min. Remove the supernatant. **Resuspended the pellet with 1 mL cold PBS. Carefully repeat pipetting for ~20 times. '''Note: It is suggested to take the tube out of foil for this resuspending step, in which it's easy to see the complete suspension and prevent the pellet loss by sticking to the tips. **Pellet labeled bacteria again with 8000 x g, 3 min. Remove the supernatant (pour + p10 tips). '''NOTE: The pellet of 100uL-sample is too small after 8000xg, 3min centrifuge. Another 14000xg, 3min centrifuge was applied to get the sufficient pellet.''' **Resuspended the pellet with PBS. According to the pellet size. 1mL-sample pellet was resuspended with 200uL PBS. 100uL-sample was resuspended with 50uL PBS. Store samples immediately in dark place at 4C.
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