Editing
Matt:LabNotes/Probe Production and Capture
(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!
===Production PCR=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''32x rxn mix''' |- | 1st round amplicons (10nM)||0.20||6.40 |- | eMIP_CA1_F (100uM)||0.40||12.80 |- | eMIP_CA1_R (100uM)||0.40||12.80 |- | 2x Kapa SYBG MM||50.00||1600.00 |- | H2O||49.00||1568.00 |- | Total||100.00||3200.00 |} '''Program'''<br> 95C 30sec -> (95C 5sec -> 60C 30sec-> 72C 20sec) x '''13''' -> 72C 2min -> 15C hold * Monitor cycles to prevent overamplification * Check the oligos by running 2ul before amplification in 6% TBU gel and quantify conc. if it corresponds to conc. specified on the tube? * Perform EtOH precipitation of the 32 rxn PCR in 4x 15mL tube ** 800ul amplified amplicon +80ul 3M NaOAc+2.6ul Glycoblue and 2mL 100EtOH ** incubate at -80C for 30min ** centrifuge at 3,000 rpm for 30 min at 4C ** discard supernatant and add 800ul cold 75% EtOH ** transfer DNA pellet to 1.5 mL tube (using pre-cut 1mL pipette tip) ** spin at 14,000 rpm for 5 min ** discard supernatant and air-dry pellet for 5 min in hood ** resuspend DNA with 150ul H20 ** measure conc. with Nanodrop:__ *Re-purify amplified-amplicon with Qiaquick column? ** Pool purified amplicons into one tube and measure DNA conc with Nanodrop:__
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