Editing
Noi/NOTES/2012-8-23
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!
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] = Bisulfite conversion = * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-8-22 * Repeat bisulfite conversion and I will fill more info and reason of repeating {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Lab IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 2000ng''' | align="center" style="background:#f0f0f0;"|'''LabID ID''' | width="50pt" align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | GL-1||295.8||6.76||GL-1||13.24||2000.00||130.00||150.00 |- | GL-2||220.6||9.07||GL-2||10.93||2000.00||130.00||150.00 |- | GL-3||149.7||13.36||GL-3||6.64||2000.00||130.00||150.00 |- | GL-4||172.3||11.61||GL-4||8.39||2000.00||130.00||150.00 |- | GL-5||285.2||7.01||GL-5||12.99||2000.00||130.00||150.00 |- | GL-6||296.6||6.74||GL-6||13.26||2000.00||130.00||150.00 |- | GL-7||320.0||6.25||GL-7||13.75||2000.00||130.00||150.00 |- | GL-8||339.3||5.89||GL-8||14.11||2000.00||130.00||150.00 |- | GL-9||467.5||4.28||GL-9||15.72||2000.00||130.00||150.00 |- | GL-10||431.7||4.63||GL-10||15.37||2000.00||130.00||150.00 |- | GL-11||349.1||5.73||GL-11||14.27||2000.00||130.00||150.00 |- | GL-12||234.5||8.53||GL-12||11.47||2000.00||130.00||150.00 |- | GL-13**||72.2||20.00||GL-13||0.00||1444.80||130.00||150.00 |- | GL-14||137.3||14.56||GL-14||5.44||2000.00||130.00||150.00 |- | GL-15||207.5||9.64||GL-15||10.36||2000.00||130.00||150.00 |- | GL-16||135.5||14.76||GL-16||5.24||2000.00||130.00||150.00 |- | AL-1||226.6||8.83||AL-1||11.17||2000.00||130.00||150.00 |- | AL-2||375.5||5.33||AL-2||14.67||2000.00||130.00||150.00 |- | AL-3||256.8||7.79||AL-3||12.21||2000.00||130.00||150.00 |- | AL-4||142.1||14.08||AL-4||5.92||2000.00||130.00||150.00 |- | AL-5||140.1||14.27||AL-5||5.73||2000.00||130.00||150.00 |} * Note: Sample GL-5, GL-7 and GL-8 have lower yield than other samples, so I did bis-cvt in duplicate '''Program''' 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold * Elute with 30ul Elution buffer, after Qubit quantification I have ~25ul for BSPP capture == 330k BSPP capture set up == === Probe calculation === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Subset ID''' | align="center" style="background:#f0f0f0;"|'''Adjusted Multiplier(2012_05_22)''' | align="center" style="background:#f0f0f0;"|'''Actual amount of probe required (probe:target ratio, 100:1)''' | align="center" style="background:#f0f0f0;"|'''Multiplier F''' | align="center" style="background:#f0f0f0;"|'''Probe concentration (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 23rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''Subset ID''' |- | DMR.s1||0.25||2.24||0.56||26.2||0.02||0.49||DMR.s1 |- | DMR.s2||0.35||2.24||0.78||30.1||0.03||0.60||DMR.s2 |- | DMR.s3||1.15||18.90||21.74||21.15||1.03||23.64||DMR.s3 |- | DMR.s4||1.00||18.90||18.90||22.34||0.85||19.46||DMR.s4 |- | DMR.s5||4.83||18.90||91.29||29||3.15||72.40||DMR.s5 |- | DMR.Exp1||1.00||18.90||18.90||31.88||0.59||13.64||DMR.Exp1 |- | DMR.Exp2||1.00||18.90||18.90||24.11||0.78||18.03||DMR.Exp2 |- | DMR.Exp3||1.00||18.90||18.90||28.9||0.65||15.04||DMR.Exp3 |- | 1.05uM Oligo suppressor-set1||||||||||0.125||2.88||1.05uM Oligo suppressor-set1 |- | 1.05uM Oligo suppressor-set2||||||||||0.125||2.88||1.05uM Oligo suppressor-set2 |} * This means that the maximum volume of bis-cvt gDNA is 42.15ul in total volume 55ul === Reaction mix === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''1rxn (ul)''' | align="center" style="background:#f0f0f0;"|'''23 rxn mix (ul)''' |- | 10X AmpLigase Buffer||5.50||126.50 |- | DMR330k probe mix (ul)||7.10||163.30 |- | 105nM Oligo suppressor||0.25||5.75 |- | Total volume (ul)||12.85||295.55 |} <br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Volume for 250ng (ul)''' | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total amount of<br> bis-cvt gDNA (ng)''' | align="center" style="background:#f0f0f0;"|'''Probe mix (ul)''' | align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | align="center" style="background:#f0f0f0;"|'''1.05uM Oligo suppressor<br> set1 &2 (ul)''' | align="center" style="background:#f0f0f0;"|'''Total volume (ul)''' |- | GL-1||6.30||39.68||GL-1||2.47||250.00||7.10||5.50||0.25||55.00 |- | GL-2||5.00||42.15||GL-2||0.00||210.75||7.10||5.50||0.25||55.00 |- | GL-3||8.92||28.03||GL-3||14.12||250.00||7.10||5.50||0.25||55.00 |- | GL-4||7.14||35.01||GL-4||7.14||250.00||7.10||5.50||0.25||55.00 |- | GL-5*||5.34||42.15||GL-5*||0.00||225.08||7.10||5.50||0.25||55.00 |- | GL-6##||3.97||42.15||GL-6##||0.00||167.34||7.10||5.50||0.25||55.00 |- | GL-7*||5.30||42.15||GL-7*||0.00||223.40||7.10||5.50||0.25||55.00 |- | GL-8*||5.08||42.15||GL-8*||0.00||214.12||7.10||5.50||0.25||55.00 |- | GL-9||5.51||42.15||GL-9||0.00||232.25||7.10||5.50||0.25||55.00 |- | GL-10||5.31||42.15||GL-10||0.00||223.82||7.10||5.50||0.25||55.00 |- | GL-11||4.99||42.15||GL-11||0.00||210.33||7.10||5.50||0.25||55.00 |- | GL-12||4.97||42.15||GL-12||0.00||209.49||7.10||5.50||0.25||55.00 |- | GL-13||7.11||35.16||GL-13||6.99||250.00||7.10||5.50||0.25||55.00 |- | GL-14||7.27||34.39||GL-14||7.76||250.00||7.10||5.50||0.25||55.00 |- | GL-15||8.01||31.21||GL-15||10.94||250.00||7.10||5.50||0.25||55.00 |- | GL-16||7.25||34.48||GL-16||7.67||250.00||7.10||5.50||0.25||55.00 |- | AL-1||5.06||42.15||AL-1||0.00||213.28||7.10||5.50||0.25||55.00 |- | AL-2||5.00||42.15||AL-2||0.00||210.75||7.10||5.50||0.25||55.00 |- | AL-3||5.22||42.15||AL-3||0.00||220.02||7.10||5.50||0.25||55.00 |- | AL-4||7.16||34.92||AL-4||7.23||250.00||7.10||5.50||0.25||55.00 |- | AL-5||8.40||29.76||AL-5||12.39||250.00||7.10||5.50||0.25||55.00 |} '''Program''' -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (08.24.2012, ~5pm) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. === SLN mix solution === {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Stock conc.''' | width="40pt" align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Final conc.''' | width="40pt" align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''Prepare volume72ul''' |- | AmpliTaq Stoffel||10||U/ul||2||U/ul||14.40 |- | AmpLigase||5||U/ul||0.5||U/ul||7.20 |- | dNTP||1000||uM||100||uM||7.20 |- | 10x AmpLigase Buffer||10||x||1||x||7.20 |- | H2O||||||||||36.00 |- | Total||||||||||72.00 |} * Add 3ul of SLN mix to each '''2012.08.25'''<br> == PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 Ind9-29) == * I tested if the capture works well by performing qPCR of few samples in small volume 25ul * Sample te be tested: NTC, GL7,GL8 AL-1 and AL3 * Since the total volume of capture reaction is large (55ul), I will add more template in the PCR reaction to the previous experiment (~20% V/V -> 5ul in 25ul reaction) {| {{table}} border =1 | align="center" style="background:#f0f0f0;"|'''Components''' | width="55pt" align="center" style="background:#f0f0f0;"|'''1 rxn''' | width="80pt" align="center" style="background:#f0f0f0;"|'''5.2x rxn mix''' |- | Circularized DNA template||5.00||0.00 |- | 10uM AmpF6.4Sol||0.50||2.60 |- | 10uM AmpR6.3Sol||0.50||2.60 |- | 50x SYBG||0.20||1.04 |- | 2x Phusion MM||12.50||65.00 |- | H2O||6.30||32.76 |- | Total||25.00||104.00 |} * Aliquot 20ul, add 5ul of DNA template '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min [[File:20120825_testqPCR_BSPP-GL-AL.png| 600px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | width="60pt" align="center" style="background:#f0f0f0;"|'''1 rxn''' | width="80pt" align="center" style="background:#f0f0f0;"|'''45x rxn mix''' |- | Captured template||20.00||0.00 |- | 10uM AmpF6.4Sol||2.00||90.00 |- | 10uM AmpR6.3Indx (X=9-29)||2.00||0.00 |- | 50x SYBG||0.80||36.00 |- | 2x Phusion MM||50.00||2250.00 |- | H2O||25.20||1134.00 |- | Total||100.00||3510.00 |} '''Program''' 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x11 -> 72C 3min (All reactions were stopped at cycle 19) [[File:20120825_qPCR_BSPP-GL-AL.png| 600px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Indx''' | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Indx''' | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | GL-1||AmpR6.3Indx9||GL-9||AmpR6.3Indx17||AL-1||AmpR6.3Indx25 |- | GL-2||AmpR6.3Indx10||GL-10||AmpR6.3Indx18||AL-2||AmpR6.3Indx26 |- | GL-3||AmpR6.3Indx11||GL-11||AmpR6.3Indx19||AL-3||AmpR6.3Indx27 |- | GL-4||AmpR6.3Indx12||GL-12||AmpR6.3Indx20||AL-4||AmpR6.3Indx28 |- | GL-5||AmpR6.3Indx13||GL-13||AmpR6.3Indx21||AL-5||AmpR6.3Indx29 |- | GL-6||AmpR6.3Indx14||GL-14||AmpR6.3Indx22|||| |- | GL-7||AmpR6.3Indx15||GL-15||AmpR6.3Indx23|||| |- | GL-8||AmpR6.3Indx16||GL-16||AmpR6.3Indx24|||| |} - Took 80ul of PCR product from replicate1 and 2 (total volume 160ul) - Purified with 0.8 vol of AmPure beads (I diluted AMPure bead 1:1 ratio with 20%PEG/2.5M NaCl) - Eluted with 60ul EB buffer - Performed PAGE quantification by loading sample 1ul [[File:ZhangLab_2 2012-08-25 21hr 25min_PQ_GL-1.jpg| 500px]] [[File:ZhangLab_2 2012-08-25 21hr 25min_PQ_GL-2.jpg| 500px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields in 60ul (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume for 200ng (ul)''' |- | GL-1||10.72||642.91||18.67 |- | GL-2||12.24||734.53||16.34 |- | GL-3||11.98||718.56||16.70 |- | GL-4||12.17||730.11||16.44 |- | GL-5||11.07||664.39||18.06 |- | GL-6||12.74||764.43||15.70 |- | GL-7||10.40||623.89||19.23 |- | GL-8||9.74||584.46||20.53 |- | GL-9||10.88||652.62||18.39 |- | GL-10||11.16||669.83||17.92 |- | GL-11||8.30||497.79||24.11 |- | GL-12||9.40||564.06||21.27 |- | GL-13||9.26||555.74||21.59 |- | GL-14||7.18||430.56||27.87 |- | GL-15||7.37||441.94||27.15 |- | GL-16||8.25||494.83||24.25 |- | AL-1||5.11||306.64||39.13 |- | AL-2||6.38||382.88||31.34 |- | AL-3||5.96||357.37||33.58 |- | AL-4||4.91||294.31||40.77 |- | AL-5||4.45||266.78||44.98 |} - Combine 200ng of each library. -Perform PAGE size selection in 6% TBE 5-well gel (3 gels) [[File:ZhangLab_2 2012-08-26 18hr 46min_PAGE_SS-1.jpg| 500px]] [[File:ZhangLab_2 2012-08-26 18hr 29min_PAGE_SS-2.jpg| 250px]] * From gel images, DNA was overoaded - Resuspended with H2O, total volume 48ul [[File:ZhangLab_2 2012-08-26 22hr 24min_PQ_NP_BSPP_GL-AL.Ind9-29.jpg| 200px]] == dsDNA Qubit HS quantification == {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Library IDs''' | align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit''' | align="center" style="background:#f0f0f0;"|'''Unit''' | align="center" style="background:#f0f0f0;"|'''uL used''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Ave. Conc. (ng/ul)''' |- | AL-exome_Aug21 1ul||46.60||ng/mL||1.00||200||9.32||9.76 |- | AL-exome_Aug21 2ul||102.00||ng/mL||2.00||100||10.20|| |- | NP_WGBS_N37_Aug26 1ul||18.70||ng/mL||1.00||200||3.74||3.80 |- | NP_WGBS_N37_Aug26 2ul||38.50||ng/mL||2.00||100||3.85|| |- | NP-BSPP-GL-AL_Ind9-29_Aug23 1ul||72.50||ng/mL||1.00||200||14.50||14.95 |- | NP-BSPP-GL-AL_Ind9-29_Aug23 2ul||154.00||ng/mL||2.00||100||15.40|| |} <br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Library IDs''' | align="center" style="background:#f0f0f0;"|'''Ave. Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Size (bp)''' | align="center" style="background:#f0f0f0;"|'''Conc. (nM)''' | align="center" style="background:#f0f0f0;"|'''MW (g/mole)''' |- | AL-exome_Aug21||9.76||300||53.52||182,377.9 |- | NP_WGBS_N37_Aug26||3.80||500||12.49||303,857.9 |- | NP-BSPP-GL-AL_Ind9-29_Aug23||14.95||375||65.59||227,932.9 |} - Quantified concentration by Qubit dsDNA assay HS (based on size 375bp): 14.95ng/ul or 65.59nM - Concentration by PAGE quantification is 26.88ng/ul * '''Library IDs: NP-BSPP-GL-AL_Ind9-29_Aug23, 2012, sequenced on HiSeqAug28 run at Biogem (Lane : 7-8)'''
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)
Template used on this page:
Template:Table
(
edit
)
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