Editing
Noi/NOTES/2011-10-6
(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!
== Amplification == * To test whether the capture work and what is the best annealing temperature for this probe set, I ran the PCR in small volume 25ul and using 2ul of circularized DNA template. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1 rxn''' | align="center" style="background:#f0f0f0;"|'''14rxn mix''' |- | Captured DNA template||2.00||0.00 |- | 10uM CP-2-FA||0.50||7.00 |- | 10uM CP-2-RA||0.50||7.00 |- | 2xKAPA MM||12.50||175.00 |- | H2O||9.50||133.00 |- | Total||25.00||350.00 |} * Aliquot 23ul, add 2ul of DNA template '''Program'''<br> 98C 30sec--> (98C 10sec --> 52C 30sec --> 72C 30sec)x5 cycles --> (98C 10sec --> 60C 30sec --> 72C 30sec)x15 cycles--> 72C 3 min --> 15C hold<br> [[File:2011_10_10-test capture20cycles.png| 700px]]<br> {| {{table}} | align="center" style="background:#f0f0f0;"|'''Samples''' | align="center" style="background:#f0f0f0;"|'''Ct 48C''' | align="center" style="background:#f0f0f0;"|'''Ct 50C''' |- | PGP1F||16.77||16.92 |- | PGP1iPS||16.72||17.12 |- | H1||16.51||16.81 |}<br> * To verify the PCR product, 2 ul of each samples were loaded in 6% TBE gel [[File:ZhangLab_2 2011-10-10 17hr 30min_Amp-CP-2.jpg| 500px]] * The expected band of amplicon ~ 260bp. Since there were many bands appeared in the gel, Dr. Zhang suggested to do PAGE size-selection to cut the 260bp band before doing PCR with the barcoded primers. * I repeated the amplification again in total volume 100ul by using the circularized DNA from 50, 52 and 54C annealing temperature and purified with Ampure beads (eluted volume 50ul). However, I used only the samples from 52 and 54C in PAGE size-selection and the next step. [[File:ZhangLab_2 2011-10-11 00hr 03min-PAGE-SS-PGP1F.jpg| 320px]][[File:ZhangLab_2 2011-10-10 23hr 57min-PAGE-SS-PGP1iPS.jpg| 320px]] [[File:ZhangLab_2 2011-10-10 23hr 55min-PAGE-SS-H1.jpg| 320px]]<br> * After EtOH precipitation I resuspended each samplle with 12ul H2O.
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