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!
=== PCR in large volume set up === ==== IMPORTANT NOTE: ==== * We try to amplified every captured molecules, so at least 2/3 of captured reaction should be amplified by PCR. * We can use any proofreading DNA polymerase for amplification such as 2XPhusion® High-Fidelity PCR Master Mix from NEB or 2X KAPA HiFi HotStart ReadyMix from KAPABiosystem. We do not use regular Taq-based DNA polymerase to avoid strand displacement during amplification and it will result in large amplified amplicons. <br> {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''11.5x rxn''' |- | Captured template||20.00||0.00 |- | 10uM AmpF6.4Sol||4.00||46.00 |- | 10uM AmpR6.3.IndX 8||4.00||0.00 |- | 2X Phusion Flash MM||100.00||1,150.00 |- | 50X SYBR||1.60||18.40 |- | H2O||70.40||809.60 |- | Total||200.00|| |} :- Aliquot 176ul + 4ul of 10uM AmpR6.3 IndXX + 20ul template (avoid pipetting mineral oil, I always spin down the tube before pipetting) :- Mix well and split into 2X of 100ul :: ''98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min (total 19 cycles)'' * Use the same condition as in quick test PCR, but amplify in larger volume (prepare 200ul PCR/sample --> split to 2X of 100ul reaction with indexing barcode primers, reverse primer) # In 100ul PCR reaction, it consists of 10 μL of circularized DNA, 200 nM each of AmpF6.4Sol primers and AmpR6.3 indexing primer, and 1x Phusion Flash mastermix. # The PCR programs of Phusion Flas are 98 ºC for 30 s, 8 cycles of 98 ºC for 10 s; 58 ºC for 20 s; and 72 ºC for 20 s, 10-15 cycles of 98 ºC for 10 s and 72 ºC for 20 s, and final extension at 72 ºC for 3 minutes. Monitor qPCR during amplification and stop the reaction before the fluorescent curve reaches plateau to prevent over-amplification. # Verify the amplified amplicons in 12-well 6% TBE gel by loading 3 μL of PCR products and run at 250 V in 0.5x TBE buffer for 25 minutes. # Stain the gel with 1x SYBR Gold in 0.5x TBE buffer for 4 minutes and visualize in Gel Doc. # Pool 80ul of PCR product from each tube (total 160ul) and purified PCR products with 0.8x volume of AMPure magnetic beads using standard protocol by the manufacturer and elute with 50 μL TE buffer. # Measure concentration of purified PCR products with Bioanalyzer ## I took 3ul of bead purified sequencing libraries, but made some mistake by mixing with non-purified PCR product. I did not have gel image of bead purified sequencing libraries. ## I handed all bead purified sequencing libraries to Li for BA measurement and sequencing # Pool equimolar ratio of each library into single tube for PAGE size selection if there are high background in the libraries after bead purification. If the band is clear, we can easily re-purify with 0.8X AMPure bead and resuspend with small volume of TE buffer to increase concentration of library pool. [[File:Beadpurified-BSPP-BA-gel.png| 650px]] [[File:Beadpurified-BSPPLib.jpg| 600px]]
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