Editing
Noi/NOTES/2014-6-16
(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 == === TEST PCR === {| {{table}} border = 1 | align="center" style="width:160px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''13 rxn mix''' |- | Bis-cvt DNA||||||||||6.00||0.00 |- | 10X Reaction buffer||10||X||1||X||2.00||26.00 |- | dNTP mix||10||mM||0.25||mM||0.50||6.50 |- | TruS_F/R||10||uM||0.3||uM||0.60||7.80 |- | 50X SYBG||50||X||0.8||X||0.32||4.16 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||5.20 |- | H2O||||||||||10.18||132.34 |- | Total ||||||||||20.00|| |} :- Aliquot 14 + 6ul DNA template :- Mix well. Split 2X of 9ul :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X [15X & 17X] --> 72C for 2min'' :- Load 5ul PCR product in 12% gel and run at 250 volts for 40min [[File:ZhangLab_2 2014-06-20 15hr 53min_PC-T_RRBS_15X_TEST.jpg|490px]] [[File:ZhangLab_2 2014-06-20 21hr 11min_PC-T_RRBS_17x_TEST.jpg|500px]] - I reloaded 17X cycles gel. The intensity of 15X and 17X cycles were not much different because I had ~4ul left not exactly 5ul as labeled in the gel image. - The signal in NTC should come from increasing amount of spike-in lambda DNA to 1ng. * Then set up reaction 100ul for each samples with 30ul BIS-DNA and split into 2X of 50ul to avoid bias from amplification (Ref. split 4X of 25ul) * Do not include NTC if NTC is clean in TEST PCR {| {{table}} border = 1 | align="center" style="width:160px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc./amount''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''10.5 rxn mix''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||10.00||105.00 |- | dNTP mix||10||mM||0.25||mM||2.50||26.25 |- | TruS_F/R||10||uM||0.3||uM||3.00||31.50 |- | 50X SYBG||50||X||0.8||X||1.60||16.80 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||2.00||21.00 |- | H2O||||||||||50.90||534.45 |- | Total ||||||||||100.00|| |} :- Aliquot 70 + 30ul DNA template :- Mix well. Split 2X of 50ul (actual ~48ul) :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 15 cycles --> 72C for 2min'' :- Stopped at 15X cycles based on TEST PCR :- I pooled the two PCR replicatea (total ~90ul for each samples) and purified with 1x volume AMPure beads and eluted with 50ul EB buffer :- I did PAGE quantification by loading ul of bead-purified sequencing libraries [[File:ZhangLab_2 2014-06-20 21hr 08min_PC-T_100ngRRBS_PQ_3ul_e.jpg| 510px]] ==== PAGE quantification results ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:60px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 47ul (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 200ng (ul)''' |- | PC-T-1_1||1||6.37||299.43||31.39 |- | PC-T-1_2||2||4.55||213.68||43.99 |- | PC-T-2_1||4||15.85||744.80||12.62 |- | PC-T-2_2||5||7.27||341.89||27.49 |- | PC-T-4_1||6||6.77||318.11||29.55 |- | PC-T-4_2||7||6.18||290.67||32.34 |- | PC-T-6_1||8||13.18||619.39||15.18 |- | PC-T-6_2||12||10.99||516.72||18.19 |- | PC-T-7_1||14||5.49||258.14||36.41 |- | PC-T-7_2||15||5.70||267.95||35.08 |} * The concentration is quite over-estimated. However, all samples were quantified in the same way and on the same gel. It should not affect the molar ratio of sequencing libraries in the pool. :- I pooled 200ng each library based on PAGE quantification and did size selection in 6 well of 5-well 6% TBE gel<br> [[File:ZhangLab_2 2014-06-22 11hr 20min_PC-T_rrbs-100ng_PAGE-SS-1.jpg| 400px]] [[File:ZhangLab_2 2014-06-22 11hr 20min_PC-T_rrbs-100ng_PAGE-SS-2.jpg| 390px]] :- I precipitated in 6tubes at -80C O/N and resuspended each tube with 11ul H2O --> total volume 66ul :- I took 4ul for PAGE verification ==== PAGE verification ==== [[File:ZhangLab_2 2014-06-24 17hr 06min_RRBS_PAGE-verify.jpg| 500px]] '''Sequencing library ID: NP-RRBS-PCT-100ng-Jun16, 2014'''<br> - Length: 160-360bp<br> ==== <span style="color:blue"><u>Index for 100ng tumor tissue RRBS sequencing libraries</u></span> ==== {| {{table}} class = wikitable | align="center" style="width:120px;background:#f0f0f0;"|'''Project ID''' | align="center" style="background:#f0f0f0;"|'''Sample type''' | align="center" style="background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' |- | UCSD-004-04||Solid tumor tissue||PC-T-1_1||1 |- | ||Solid tumor tissue||PC-T-1_2||2 |- | UCSD-004-05||Solid tumor tissue||PC-T-2_1||4 |- | ||Solid tumor tissue||PC-T-2_2||5 |- | UCSD-004-07||Solid tumor tissue||PC-T-4_1||6 |- | ||Solid tumor tissue||PC-T-4_2||7 |- | UCSD-004-09||Solid tumor tissue||PC-T-6_1||8 |- | ||Solid tumor tissue||PC-T-6_2||12 |- | UCSD-004-10||Solid tumor tissue||PC-C-7_1||14 |- | ||Solid tumor tissue||PC-C-7_2||15 |}
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