Editing
Noi/NOTES/2014-8-15
(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!
= Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair) = [[noi:DMR220k_LabNotes|'''Back to calendar''']] {| {{table}} border =1 | align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 1ng''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O''' | align="center" style="width:60px;background:#f0f0f0;"|'''# in strip''' | align="center" style="width:80px;background:#f0f0f0;"|'''Exp ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Klenow,exo-:dAdCdGdT Mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total''' |- | NC-1||0.293||3.41||6.59||#1_1||wNC-1||10.00||20.00 |- | NC-2||0.325||3.08||6.92||#1_2||wNC-2||10.00||20.00 |- | NC-3||0.538||1.86||8.14||#1_3||wNC-3||10.00||20.00 |- | NC-5||0.156||6.41||3.59||#1_4||wNC-5||10.00||20.00 |- | NC-6||0.148||6.76||3.24||#1_5||wNC-6||10.00||20.00 |- | NC-7||0.245||4.08||5.92||#1_6||wNC-7||10.00||20.00 |- | NC-8||0.141||7.09||2.91||#1_7||wNC-8||10.00||20.00 |- | NC-9||0.171||5.85||4.15||#1_8||wNC-9||10.00||20.00 |- | NC-12||0.163||6.13||3.87||#2_1||wNC-12||10.00||20.00 |- | NC-13||0.168||5.95||4.05||#2_2||wNC-13||10.00||20.00 |- | NC-14||0.157||6.37||3.63||#2_3||wNC-14||10.00||20.00 |- | NC-15||0.118||8.47||1.53||#2_4||wNC-15||10.00||20.00 |- | NC-16||0.140||7.14||2.86||#2_5||wNC-16||10.00||20.00 |- | NC-17||0.126||7.94||2.06||#2_6||wNC-17||10.00||20.00 |- | NC-18||0.189||5.29||4.71||#2_7||wNC-18||10.00||20.00 |- | NC-19||0.105||9.52||0.48||#2_8||wNC-19||10.00||20.00 |- | NC-20||0.128||7.81||2.19||#3_1||wNC-20||10.00||20.00 |- | NC-21||0.205||4.88||5.12||#3_2||wNC-21||10.00||20.00 |- | NC-22||0.180||5.56||4.44||#3_3||wNC-22||10.00||20.00 |- | NC-23||0.153||6.54||3.46||#3_4||wNC-23||10.00||20.00 |- | NC-24||0.140||7.14||2.86||#3_5||wNC-24||10.00||20.00 |- | NC-27||0.124||8.06||1.94||#3_6||wNC-27||10.00||20.00 |- | NC-29||0.109||9.17||0.83||#3_7||wNC-29||10.00||20.00 |- | NC-30||0.099||10.00||0.00||#3_8||wNC-30||10.00||20.00 |- | NTC_1||0||0.00||10.00||#4_1||||10.00||20.00 |- | NTC_2||0||0.00||10.00||#4_2||||10.00||20.00 |- | NC-3||0.538||1.86||8.14||#4_3||wNC-3t||10.00||20.00 |- | NC-4||0.848||1.18||8.82||#4_4||wNC-4t||10.00||20.00 |} * The last two samples, NC-3 and NC-4 were used to tested for T4 DNA ligase HC that has been delivered during weekend (Saturday) and was left at RT until Monday. I got the new tube from Thermoscientific for replacement. All samples except the last two were prepared with the new tube of T4 DNA ligase HC. * Prepare new tube of dA:dC:dG:dT (20:2:2:2 mM) {| {{table}} border = 1 | align="center" style="width:120px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc. (nM)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' |- | 100mM dATP||20||100 |- | 100mM dCTP||2||10 |- | 100mM dGTC||2||10 |- | 100mM dTTC||2||10 |- | H2O||||370 |} ==== <u>Klenow exo- and dA:dG:dC reaction mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''30x rxn''' |- | 10X Tango buffer||2.00||60.00 |- | dA:dC:dG:dT (20:2:2:2mM)||1.00||30.00 |- | Klenow fragment, exo- (5U/ul)||1.00||30.00 |- | H2O||6.00||180.00 |- | Total||10.00|| |} - Aliquot 32 of Klwnow, exo- and dA:dC:dG:dT reaction mix to 8-tube strip<br> :- Add H2O to each tube :- Add 1ng of DNA to each tube :- Add 10ul of Klwnow, exo- and dA:dC:dG reaction mix to each tube of DNA with multichannel pipette. Pipette few times :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 30C, 20min --> 37C, 20min --> hold at 4C (no heat lid) :- 75C, 10min --> hold at 4C (heat lid on) * No AMPure bead purification<br> <br> ==== <u>Ligation reaction mix</u> ==== {| {{table}} class = wikitable | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' | align="center" style="width:80px;background:dodgerblue;"|'''Exp ID''' | align="center" style="width:80px;background:magenta;"|'''TruSeq Index''' |- | wNC-1||1||wNC-12||13||wNC-20||11||NTC_1||3 |- | wNC-2||2||wNC-13||14||wNC-21||12||NTC_2||9 |- | wNC-3||4||wNC-14||15||wNC-22||21||wNC-3t||23 |- | wNC-5||5||wNC-15||16||wNC-23||22||wNC-4t||25 |- | wNC-6||6||wNC-16||27||wNC-24||3|||| |- | wNC-7||7||wNC-17||18||wNC-27||9|||| |- | wNC-8||8||wNC-18||19||wNC-29||23|||| |- | wNC-9||10||wNC-19||20||wNC-30||25|||| |} ==== <u>Ligation reaction mix</u> ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''28x rxn''' |- | dA-tailed DNA||20.00||0.00 |- | 10X Tango buffer||0.50||14.00 |- | HC T4 DNA ligase (30units/ul)||1.00||28.00 |- | 10mM ATP ||1.25||35.00 |- | H2O||1.25||35.00 |- | Total||24.00|| |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''1x rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''2x rxn''' |- | dA-tailed DNA||20.00||0.00 |- | 10X Tango buffer||0.50||1.00 |- | THAWED HC T4 DNA ligase (30units/ul)||1.00||2.00 |- | 10mM ATP ||1.25||2.50 |- | H2O||1.25||2.50 |- | Total||24.00|| |} :- Aliquot 13ul of ligation reaction mix to 8-tube strip :- Add 1ul of 1:20 diluted TruSeq adaptors and pipette few times to make sure adaptors were added to the reaction :- Add 4ul of ligation reaction mix with multichannel pipette :- Spin down the tube :- Mix by gentle pulse-vortexing on PCR rack 10x :- Spin down the tube :- 16C, 20h (no heat lid) :- Heat inactivate at 65C, 20min --> hold at 4C (heat lid on) :- Continue to bisulfite conversion == Bisulfite conversion == [[Noi/NOTES/2014-8-3#Bisulfite_conversion| '''Bisulfite conversion procedures''']]<br> == Amplification == * Repeat all procedure exactly the same as previous experiment on 2014-08-03 === 1st round PCR (fix 12 cycles) === {| {{table}} border =1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;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)''' |- | Bis-cvt DNA||||||||||30.00 |- | 10X Reaction buffer||10||X||1||X||5.00 |- | dNTP mix||10||mM||0.25||mM||1.25 |- | TruS_F/R||10||uM||0.3||uM||1.50 |- | 50X SYBG||50||X||0.4||X||0.40 |- | PfuTurbo Cx||2.5||Unit/ul||1||unit||1.00 |- | H2O||||||||||10.85 |- | Total ||||||||||50.00 |} :- Aliquot 20 + 30ul BIS-DNA template :- Mix well :: ''95C for 2min --> [95C for 20sec -> 60C for 30sec -> 72C for 1min] X 12 --> 72C for 2min'' :- Purify with 1.25X AMPure beads :- Elute with 42ul EB Buffer :- Continue to 2nd round PCR. No PAGE verification. === 2nd round PCR === === Quick test === {| {{table}} border =1 | align="center" style="width:140px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc''' | align="center" style="width:80px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Final conc.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''30 rxn mix''' |- | Purified 1st round DNA||||||||||3.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||2.00||60.00 |- | dNTP mix||10||mM||0.25||mM||0.25||7.50 |- | TruS_F/R||10||uM||0.3||uM||0.30||9.00 |- | 50X SYBR||50||X||0.4||X||0.08||2.40 |- | Phusion HF||2||unit/ul||||||0.10||3.00 |- | H2O||||||||||4.27||128.10 |- | Total||||||||||10.00|| |} :- Aliquot 7 + 3ul DNA template :- Mix well. ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 8 cycles --> 72C for 2min''<br> :- Added 2ul 6X loading dye to each strip tube & mix well :- Load 6ul PCR/dye mix in 6% gel and run at 250 volts for 22min. [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel1.jpg|450px]] [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel2.jpg|450px]] [[File:ZhangLab_2 2014-08-17 16hr 58min_test-2ndPCR_NC-8x_gel3.jpg|430px]] * From quick qPCR test, there are variation of the intensity of libraries size ~300bp. 8 cycles seem to be over-amplified for almost samples. There are some samples that had very faint intensity of the libraries. I need to varied PCR cycle number for some samples. {| {{table}} border = 1 | align="center" style="width:140px;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.''' | align="center" style="width:60px;background:#f0f0f0;"|'''unit''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''27 rxn mix''' |- | Purified 1st round DNA||||||||||36.00||0.00 |- | 5X Phusion HF buffer||5||X||1||X||24.00||648.00 |- | dNTP mix||10||mM||0.25||mM||3.00||81.00 |- | TruS_F/R||10||uM||0.3||uM||3.60||97.20 |- | 50X SYBR||50||X||0.4||X||0.96||25.92 |- | Phusion HF||2||unit/ul||||||1.20||32.40 |- | H2O||||||||||51.24||1383.48 |- | Total||||||||||120.00|| |} :- Aliquot 84 + 36ul DNA template :- Mix well. Split 38ul X3 ::''98C for 2min --> [98C for 10sec -> '''60C for 30sec''' -> 72C for 1min] X 7 cycles --> 72C for 2min''<br> :- I pool ~110ul PCR product from the two replicates, purify with 2 rounds of 1X AMPure beads (fist round elute with EB buffer 60ul. 2nd round elute with 65ul EB buffer. :- Loaded 2ul in 6% TBE gel for PAGE verification [[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel1.jpg| 450px]] [[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel2.jpg| 450px]] [[File:ZhangLab_2 2014-08-18 14hr 52min_PQ_wgbs_NC-2ul_gel3.jpg| 320px]] * Different from WGBS from cancer patient small fragment DNA, there are high variations of library amount among different samples as mentioned from quick qPCR. Also, the last two samples (NC-3 and NC-4) ligated with T4 DNA ligase left at RT almost 2 days had comparable result compare to the same sample ligated with proper stored T4 DNA ligase. === Qubit dsDNA HS quantification of WGBS libraries from normal control cfDNA === {| {{table}} border =1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. in the Qubit''' | align="center" style="width:60px;background:#f0f0f0;"|'''Unit''' | align="center" style="width:60px;background:#f0f0f0;"|'''uL used''' | align="center" style="width:60px;background:#f0f0f0;"|'''Dilution''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Used for prev. exp (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount left (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''> 400ng''' |- | NC-1||4.1||ng/mL||2||100||0.41||80.00||32.80||0.00||32.80||NO |- | NC-2||13.9||ng/mL||2||100||1.39||80.00||111.20||0.00||111.20||NO |- | NC-3||18||ng/mL||2||100||1.80||80.00||144.00||0.00||144.00||NO |- | NC-5||4.71||ng/mL||2||100||0.47||80.00||37.68||0.00||37.68||NO |- | NC-6||42.5||ng/mL||2||100||4.25||80.00||340.00||100.00||240.00||NO |- | NC-7||4.69||ng/mL||2||100||0.47||80.00||37.52||0.00||37.52||NO |- | NC-8||15.2||ng/mL||2||100||1.52||80.00||121.60||0.00||121.60||NO |- | NC-9||37.2||ng/mL||2||100||3.72||80.00||297.60||100.00||197.60||NO |- | NC-12||29.9||ng/mL||2||100||2.99||80.00||239.20||0.00||239.20||NO |- | NC-13||16.4||ng/mL||2||100||1.64||80.00||131.20||0.00||131.20||NO |- | NC-14||12.8||ng/mL||2||100||1.28||80.00||102.40||0.00||102.40||NO |- | NC-15||12.9||ng/mL||2||100||1.29||80.00||103.20||0.00||103.20||NO |- | NC-16||13.3||ng/mL||2||100||1.33||80.00||106.40||0.00||106.40||NO |- | NC-17||38.6||ng/mL||2||100||3.86||80.00||308.80||0.00||308.80||NO |- | NC-18||26.3||ng/mL||2||100||2.63||80.00||210.40||0.00||210.40||NO |- | NC-19||4.74||ng/mL||2||100||0.47||80.00||37.92||0.00||37.92||NO |- | NC-20||3.96||ng/mL||2||100||0.40||80.00||31.68||0.00||31.68||NO |- | NC-21||4.13||ng/mL||2||100||0.41||80.00||33.04||0.00||33.04||NO |- | NC-22||27.1||ng/mL||2||100||2.71||80.00||216.80||0.00||216.80||NO |- | NC-23||17.6||ng/mL||2||100||1.76||80.00||140.80||0.00||140.80||NO |- | NC-24||3.87||ng/mL||2||100||0.39||80.00||30.96||0.00||30.96||NO |- | NC-27||68.8||ng/mL||2||100||6.88||80.00||550.40||100.00||450.40||YES |- | NC-29||35.9||ng/mL||2||100||3.59||80.00||287.20||0.00||287.20||NO |- | NC-30||64.6||ng/mL||2||100||6.46||80.00||516.80||100.00||416.80||YES |}
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