Noi/NOTES/2014-8-15: Difference between revisions
Jump to navigation
Jump to search
>Noi mNo edit summary |
>Noi mNo edit summary |
||
(7 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
= Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair) = | = 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 | {| {{table}} border =1 | ||
| align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | | align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | ||
Line 111: | Line 112: | ||
<br> | <br> | ||
==== <u>Ligation reaction mix</u> ==== | ==== <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 | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Components''' | | align="center" style="background:#f0f0f0;"|'''Components''' | ||
Line 182: | Line 210: | ||
|- | |- | ||
| Total ||||||||||50.00 | | 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 | |||
|} | |} |
Latest revision as of 18:14, 17 September 2014
Preparation of WGBS libraries of small fragment DNA in normal control plasma samples (no DNA fragmentation step, with dAdGdCdT for end repair)[edit]
Zhang lab ID | Conc. (ng/ul) | Volume for 1ng | H2O | # in strip | Exp ID | Klenow,exo-:dAdCdGdT Mix | 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)
Components | Final conc. (nM) | Volume (ul) |
100mM dATP | 20 | 100 |
100mM dCTP | 2 | 10 |
100mM dGTC | 2 | 10 |
100mM dTTC | 2 | 10 |
H2O | 370 |
Klenow exo- and dA:dG:dC reaction mix[edit]
Components | 1x rxn | 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
- - 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
Ligation reaction mix[edit]
Exp ID | TruSeq Index | Exp ID | TruSeq Index | Exp ID | TruSeq Index | Exp ID | 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 |
Ligation reaction mix[edit]
Components | 1x rxn | 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 |
Components | 1x rxn | 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[edit]
Bisulfite conversion procedures
Amplification[edit]
- Repeat all procedure exactly the same as previous experiment on 2014-08-03
1st round PCR (fix 12 cycles)[edit]
Components | Conc | unit | Final conc./amount | unit | 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[edit]
Quick test[edit]
Components | Conc | unit | Final conc. | unit | Volume (ul) | 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
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 8 cycles --> 72C for 2min
- - 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 File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel2.jpg File:ZhangLab 2 2014-08-17 16hr 58min test-2ndPCR NC-8x gel3.jpg
- 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.
Components | Conc | unit | Final conc. | unit | Volume (ul) | 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
- 98C for 2min --> [98C for 10sec -> 60C for 30sec -> 72C for 1min] X 7 cycles --> 72C for 2min
- - 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 File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel2.jpg File:ZhangLab 2 2014-08-18 14hr 52min PQ wgbs NC-2ul gel3.jpg
- 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[edit]
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Conc. (ng/ul) | Volume (ul) | Total amount (ng) | Used for prev. exp (ng) | Amount left (ng) | > 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 |