Editing
Noi/NOTES/2014-6-28
(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="bwidth:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''25 rxn mix''' |- | Bis-cvt DNA||||||||||6.00||0.00 |- | 10X Reaction buffer||10||X||1||X||2.00||50.00 |- | dNTP mix||10||mM||0.25||mM||0.50||12.50 |- | TruS_F/R||10||uM||0.3||uM||0.60||15.00 |- | 50X SYBG||50||X||0.8||X||0.32||8.00 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||0.40||10.00 |- | H2O||||||||||10.18||254.50 |- | Total ||||||||||20.00|| |} :- Aliquot 14 + 6ul DNA template :- Mix well. Split 2X of 8.5ul :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X [14X & 16X] --> 72C for 2min'' * There was error to realplex during annealing step of 15 cycles. I needed to remove samples. I loaded only 14X to save the gel and plan to loaded 16 cycles PCR if the intensity of 14X is too low. :- Add 10ul of 2X loading dye to PCR product and load 10ul PCR product/dyemix in 12% gel and run at 250 volts for 40min '''Colon tumor samples''' [[File:ZhangLab_2 2014-06-30 17hr 16min_6T_100ngRRBS_TEST-14X.jpg| 520px]] '''Lung tumor samples''' [[File:ZhangLab_2 2014-06-30 17hr 16min_7T_100ngRRBS_TEST-14X-e.jpgg| 520px]] * I do not know why sample 7-T_3.2 and 7-T_4.1 failed. May need to check with Dr. Zhang if he want to sequence the two duplicates or only 1 library for each sample. * No need to load 16 cycles PCR since 14 cycles look good and not too faint or over-amplified. * I need to repeat 7-T_3.2 and 7-T_4.1. I plan to repeat both of them in two duplicates. * Continued to prepare sequencing library by doing PCR in 100ul reaction with 30% BIS-DNA. {| {{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;"|'''20.5''' |- | Bis-cvt DNA||||||||||30.00||0.00 |- | 10X Reaction buffer||10||X||1||X||10.00||205.00 |- | dNTP mix||10||mM||0.25||mM||2.50||51.25 |- | TruS_F/R||10||uM||0.3||uM||3.00||61.50 |- | 50X SYBG||50||X||0.8||X||1.60||32.80 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||2.00||41.00 |- | H2O||||||||||50.90||1043.45 |- | 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 14 cycles --> 72C for 2min'' :- Stopped at 14X cycles based on TEST PCR :- I pooled the two PCR replicates (total ~90ul for each samples) and purified with 1x volume AMPure beads and eluted with 55ul EB buffer :- I did PAGE quantification by loading 3ul of bead-purified sequencing libraries [[File:ZhangLab_2 2014-07-02 19hr 34min_PQ_6T_100ngRRBS.jpg| 500px]] [[File:ZhangLab_2 2014-07-02 19hr 34min_PQ_7T_100ngRRBS.jpg|500px]] * It's so wired for sample 6T_4.2 since it looked fine in TEST PCR, but it's very faint after amplification in 50ul and bead purified. '''Repeat smaple 7T_4.1 (also include 7T_4.2)''' [[File:ZhangLab_2 2014-07-03 17hr 35min_PQ_repeat_7T_4.jpg| 300px]] * I reloaded sample 6T_2.1 and 6T_4.2 again. To confirm that they had very low concentration. ==== PAGE quantification results ==== <u> 006-T samples</u> {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | 6-T-1.1||1||6.53||326.73 |- | 6-T-1.2||2||9.91||495.56 |- | 6-T-2.1||23||2.00||100.20 |- | 6-T-2.2||4||7.14||356.87 |- | 6-T-3.1||5||10.60||530.01 |- | 6-T-3.2||6||6.70||334.87 |- | 6-T-4.1||7||6.19||309.49 |- | 6-T-4.2||8||0.71||35.55 |- | 6-T-5.1||25||4.31||215.60 |- | 6-T-5.2||10||8.26||412.83 |} <u> 007-T samples</u> * Value of sample 7-T-4.1 is the repeated sample (will be added) {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Index''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 50ul (ng)''' |- | 7-T-1.1||13||5.12||255.78 |- | 7-T-1.2||14||3.65||182.39 |- | 7-T-2.1||15||4.18||208.94 |- | 7-T-2.2||16||6.55||327.39 |- | 7-T-3.1||27||2.70||135.19 |- | 7-T-3.2||18||3.72||185.97 |- | <span style="color:crimson">7-T-4.1</span>||19||9.30||465.00 |- | 7-T-4.2||20||3.59||179.72 |- | 7-T-5.1||21||5.76||288.10 |- | 7-T-5.2||22||3.82||190.84 |}<br> ==== <u>Library pooling</u> ==== =====<span style="color:mediumblue"><u> 006-T samples</u></span>===== * Since I may need to sequence only one replicate, I combine sequencing libraries into two set; set 1 and set2. Set 1 set the first priority, so I included sample 6-T-2_2 in set one as sample 6-T-2_1had very faint signal. I included sample 6-T_2.1 in set 2 <span style="color:mediumblue">NP-RRBS-6T-set1_100ng-Jun28</span> {| {{table}} border = 1 | 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;"|'''Volume for 120ng (ul)''' |- | 6-T_1.1||1||6.53||18.36 |- | <span style="color:crimson">6-T_2.2</span>||4||7.14||16.81 |- | 6-T-3.1||5||10.60||11.32 |- | 6-T-4.1||7||6.19||19.39 |- | 6-T-5.1||25||4.31||27.83 |} :- Add 20ul of 6X loading dye and load 37.9ul in 3 well of 5-well 6% TBE gel <span style="color:mediumblue">NP-RRBS-6T-set2_100ng-Jun28</span> * Set 2 I include 4 libraries and do PAGE size selection of sample 6-T-4.2 separately since it had very faint smear. {| {{table}} border = 1 | 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;"|'''Volume for 100ng (ul)''' |- | 6-T-1.2||2||9.91||10.09 |- | 6-T-2.1||23||2.00||49.90 |- | 6-T-3.2||6||6.70||14.93 |- | 6-T-5.2||10||8.26||12.11 |} :- Add 20ul of 6X loading dye and load 35.68 in 3 well of 5-well 6% TBE gel <span style="color:mediumblue">NP-RRBS-6T-4_2-set2_100ng-Jun28</span> {| {{table}} border = 1 | 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;"|'''Volume (ul)''' |- | 6-T-4.2||8||0.71||50.00 |} :- Add 10ul of 6X loading dye and load 60ul in 1 well of 5-well 6% TBE gel =====<span style="color:red"><u> 007-T samples</u></span>===== <span style="color:red">NP-RRBS-7T-set1_100ng-Jun28</span> {| {{table}} border = 1 | 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;"|'''Volume for 100ng (ul)''' |- | 7-T-1.1||13||5.12||19.55 |- | 7-T-2.1||15||4.18||23.93 |- | 7-T-3.1||27||2.70||36.98 |- | 7-T-4.1||19||9.30||10.75 |- | 7-T-5.1||21||5.76||17.35 |} :- Add 25ul of 6X loading dye and load 44.52ul in 3 well of 5-well 6% TBE gel <span style="color:red">NP-RRBS-7T-set2_100ng-Jun28</span> {| {{table}} border = 1 | 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;"|'''Volume for 100ng (ul)''' |- | 7-T-1.2||14||3.65||27.41 |- | 7-T-2.2||16||6.55||15.27 |- | 7-T-3.2||18||3.72||26.89 |- | 7-T-4.2||20||3.59||27.82 |- | 7-T-5.2||22||3.82||26.20 |} :- Add 30ul of 6X loading dye and load 51.2ul in 3 well of 5-well 6% TBE gel<br> ==== PAGE size selection ==== [[File:ZhangLab_2 2014-07-06 17hr 56min_gel1.jpg|380px]] - NP-RRBS-6T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification [[File:ZhangLab_2 2014-07-06 17hr 58min_gel2.jpg|380px]] - NP-RRBS-6T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification - NP-RRBS-6T_4_2_set2_100ng-Jun28: Resuspend with 12ul H2O x 1tube--> volume 12ul, no PAGE verification [[File:ZhangLab_2 2014-07-06 18hr 03min_gel3.jpg|380px]] - NP-RRBS-7T_set1_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification [[File:ZhangLab_2 2014-07-06 18hr 04min_gel4.jpg|380px]] - NP-RRBS-7T_set2_100ng-Jun28: Resuspend with 12ul H2O x 3tubes --> volume 36ul, 4ul for PAGE verification ==== PAGE verification ==== ---- === 2014-07-08 === ==== Preparing sequencing library pool for IGM ==== * We will do HiSeq Rapid run, PE 100+7bp * Spike in with 5% PhiX in each lane :1). '''NP_RRBS_6T_100ng_20140708''' (Loaded in lane 1) :{| {{table}} class = wikitable | align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library''' | align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)''' | align="center" style="width:60px;background:#f0f0f0;"|'''Loading %''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 6T-set1''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)''' |- | NP-RRBS-6T_set1_100ng-Jun28||5||10||50||50||11.63||1||50||4.30 |- | NP-RRBS-6T_set2_100ng-Jun28||4||10||40||40||9.01||0.8||40||4.44 |- | NP-RRBS-6T_4_2_set2_100ng-Jun28||1||10||10||10||2.44||0.2||10||4.10 |- | ||||||||||||Total||100||12.84 |- | ||||||||||||Final conc.||7.79||nM |} :2). '''NP_RRBS_7T_100ng_20140708''' (Loaded in lane 2) :{| {{table}} class = wikitable | align="center" style="width:220px;background:#f0f0f0;"|'''Seq. Library''' | align="center" style="width:60px;background:#f0f0f0;"|'''# of ind. Library''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of read for each (M)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total # of reads (M)''' | align="center" style="width:60px;background:#f0f0f0;"|'''Loading %''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Ratio of % to 7T-set1''' | align="center" style="width:80px;background:#f0f0f0;"|'''# of mole to pool (nmole)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume to pool (ul)''' |- | NP-RRBS-7T_set1_100ng-Jun28||5||10||50||50||15.6||1||100||6.41 |- | NP-RRBS-7T_set2_100ng-Jun28||5||10||50||50||13.47||1||100||7.42 |- | ||||||||||||Total||200||13.83 |- | ||||||||||||Final conc.||14.46||nM |}
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