Editing
Kun:LabNotes/HiResChrPaint/2012-4-4
(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!
==Processing of oligo pools (STEP 1)== *Received the oligo pools from Mycorarray in four tubes. These are "ssDNA from emulsion PCR pool". Resuspend each pool in 500ul nuclease free H2O. *Take 50ul of each oligo pool, dilute to 20nM. These will be the working tubes. 5431_ssDNA 80k_set_4 ~2.8ug 85pmole 170nM 50ul+374ul H2O => 20nM 5432_ssDNA 80k_set_1 ~1.8ug 55pmole 109nM 50ul+223ul H2O => 20nM 5433_ssDNA 80k_set_2 ~1.7ug 52pmole 103nM 50ul+208ul H2O => 20nM 5435_ssDNA 80k_set_3 ~2.8ug 85pmole 170nM 50ul+374ul H2O => 20nM *There is probably no need to do expansion PCR since the oligo pools were already amplified by emulsion PCR. ===Confirmation PCR=== V6 pool Each 20nM oligo pool 0.4ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V61U 0.4ul 100uM AP2V6 0.4ul 2mM aa-dUTP 3ul H2O 41ul =================================== 100ul 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold. V4 pool Each 20nM oligo pool 0.4ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V41U 0.4ul 100uM AP2V4 0.4ul 2mM aa-dUTP 3ul H2O 41ul =================================== 100ul 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold. Check the size distribution of the amplicons with 6% TBE gels. [[File:4.4.12_oligopaint_1-4.jpg|400px]] [[File:4.4.12_oligopaint_5-8.jpg|400px]] [[File:4.5.12_oligocheck.jpg|400px]] ===Production PCR=== V6 pool for all four sets Each x16 20nM oligo pool 0.2ul 3.2 2x Kapa SYBG qPCR Master Mix 50ul 800 100uM pAP1V61U 0.4ul 6.4 100uM AP2V6 0.4ul 6.4 2mM aa-dUTP 3ul 48 H2O 41ul 656 =================================== 100ul 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 15 -> 72C 2min -> 15C hold. V4 pool for all four sets. Each x16 20nM oligo pool 0.2ul 3.2 2x Kapa SYBG qPCR Master Mix 50ul 800 100uM pAP1V41U 0.4ul 6.4 100uM AP2V4 0.4ul 6.4 2mM aa-dUTP 3ul 48 H2O 41ul 656 =================================== 100ul x 16 95C 30sec -> (95C 3sec -> 55C 30sec-> 72C 20sec) x 16 -> 72C 2min -> 15C hold. Check the size distribution of the amplicons with 6% TBE gels. Ethanol precipitation. Re-purified each amplicon with two Qiaquick columns, eluted with 40ul EB. Measure concentration. ===Dpn II digestion=== ===Lambda exo digestion=== ===Coupling of Alexa fluorophores with ss-DNA probes=== ===Adaptor removal=== *Dpn II digestion. ds-oligo pools 75ul 10X Dpn II buffer 10ul Dpn II (10U/ul) 8ul (might need to adjust the amount of enzyme based on the yield of PCR) H2O 7ul 37C 30min -> 65C 20min -> Qiaquick column purification *Lambda exo digestion. Dpn II digested oligo pool 40ul 10X Lambda Exo buffer 10ul Lambda Exo (10U/ul) 10ul H2O 40ul 37C 1h -> 75C 10min -> Qiaquick column purification *Proceed with AlexaFluor coupling.
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