Editing
Matt:LabNotes/2017-6-1
(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!
==Gel PCR== Prepare diffuse-in mix while allowing slides to dry. You don't want the slides to over-dry, though. You will observe a thin, shrinking film of liquid on the surface of each gel. Generally, we try to add the diffuse-in mix within 5 minutes or so of this shrinking film's disappearance. This generally means about 30 minutes of drying. #Let gel slide dry in AirClean hood for 30min #Prepare 25ul PCR mix per gel #*2.5ul 5uM Primer2 #*12.5ul KAPA SYBR FAST MM #*10ul beads (1:1 mix of v1 and v3) #Pipet 25ul onto center of gel #Apply 18x30mm cover slip #Apply an orange SecureSeal chamber #Fill chamber with mineral oil and seal holes with stickies. #Slide PCR on Biorad thermocycler 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold #Place directly on Olympus with GFP/FITC filter #*Saw polonies! #**Approx 8um in diameter and centered around the beads #**Confident not autofluorescence from the bead because only ~1/4 beads had a polony #**The 1/4 polony success rate might need to be improved but right now it may just be due to beads with no DNA #**Edit: might not be polony if SYBR Green stained dsDNA on the bead... #Put in 4C overnight
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