Editing
Noi/NOTES/2014-12-8
(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!
==== Quick PCR ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Stock conc.''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc. In PCR''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''13x rxn''' |- | Captured template||100%||10%||2.00||0.00 |- | 10uM AmpF6.4Sol||10uM||0.2uM||0.40||5.20 |- | 10uM AmpR6.3.IndX 8||10uM||0.2uM||0.40||5.20 |- | 2X Phusion Flash High-Fidelity MM||2X||1X||10.00||130.00 |- | 50X SYBR||50X||0.4X||0.16||2.08 |- | H2O||||||7.04||91.52 |- | Total||||||20.00|| |} :- Aliquot 18ul + 2ul of captured DNA template * Note: In PCR, I included one of positive control (PTC) of captured templates from gDNA of tumor tissue sample to compare the size of amplified amplicons. ::- ''98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x10-15 cycles -> 72C 3min'' :- In this experiment, we stopped at 18 cycles :- Loaded 3ul of PCR product in 6% TBE gel without purification (Run at 250 volts in 0.5X TBE buffer for 25min). [[File:ZhangLab_2 2014-12-10 15hr 14min_quickPCR-capture-18x-3ul.jpg| 500px]] [[File:ZhangLab_2 2014-12-10 15hr 14min_quickPCR-capture-18x-3ul-gel2.jpg| 250px]] PTC: Positive control NTC: Negative control I am not pretty sure what is the larger fragment is. We use enzyme without strand displacement activity, but not sure if there is some activity still exist. :- From PAGE analysis, the pattern of amplified amplicon looked similar to positive control of capture on gDNA. This is a good sign! The intensity look a little faint at 3ul loading. I will increase one more cycle (19X) for large volume amplification.
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