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>Noi (Created page with "* http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''' = DMR 330k BSPP capture of samples from Sergio = == Sample list == == New gDNA...") |
>Noi |
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* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] | * [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] | ||
= DMR 330k BSPP capture of samples from Sergio = | = DMR 330k BSPP capture of samples from Sergio = | ||
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-17 | |||
== Sample list == | == Sample list == | ||
== New gDNA extracted from the cell pellet == | == New gDNA extracted from the cell pellet == | ||
Line 149: | Line 151: | ||
== 8 samples of bis-cvt gDNA from Dinh == | == 8 samples of bis-cvt gDNA from Dinh == | ||
* The list will be filled later and need to re-measure of the conc. with ssDNA Qubit assay | * The list will be filled later and need to re-measure of the conc. with ssDNA Qubit assay | ||
= Bisulfite conversion = | |||
'''Reagent prep''' | |||
Add the following reagents to CT Conversion Reagent | |||
- H2O 9.0mL | |||
- M-Dissolving Buffer 500ul | |||
- M-Dilution Buffer 3.0mL | |||
Mix at room temperature with frequent vortexing or shaking for 15 minutes | |||
Add 144 ml of 100% ethanol to the 36 ml M-Wash Buffer concentrate before use | |||
{| {{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 1200ng''' | |||
| align="center" style="background:#f0f0f0;"|'''LabID ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Well IDs''' | |||
| 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)''' | |||
|- | |||
| Ap15-1||110.0||10.91||Ap15-1||A1||9.09||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-2||139.1||8.63||Ap15-2||B1||11.37||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-3||145.1||8.27||Ap15-3||C1||11.73||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-4||295.5||4.06||Ap15-4||D1||15.94||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-5||128.1||9.37||Ap15-5||E1||10.63||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-6||153.9||7.80||Ap15-6||F1||12.20||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-7||183.5||6.54||Ap15-7||G1||13.46||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-8||149.3||8.04||Ap15-8||H1||11.96||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-9||111.2||10.79||Ap15-9||A2||9.21||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-10||153.5||7.82||Ap15-10||B2||12.18||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-11||87.5||13.71||Ap15-11||C2||6.29||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-12||109.7||10.94||Ap15-12||D2||9.06||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-13||191.3||6.27||Ap15-13||E2||13.73||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-14||108.7||11.04||Ap15-14||F2||8.96||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-15||134.2||8.94||Ap15-15||G2||11.06||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-16||50.2||20.00||Ap15-16||H2||0.00||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-17||183.0||6.56||Ap15-17||A3||13.44||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-18||82.6||14.53||Ap15-18||B3||5.47||1200.00||130.00||150.00 | |||
|- | |||
| Ap15-19||263.4||4.56||Ap15-19||C3||15.44||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-1||86.7||13.84||Ma15-1||D3||6.16||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-2||215.2||5.58||Ma15-2||E3||14.42||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-3||252.0||4.76||Ma15-3||F3||15.24||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-4||226.1||5.31||Ma15-4||G3||14.69||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-5||238.7||5.03||Ma15-5||H3||14.97||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-6||83.4||14.39||Ma15-6||A4||5.61||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-7||236.8||5.07||Ma15-7||B4||14.93||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-8||175.9||6.82||Ma15-8||C4||13.18||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-9||164.3||7.30||Ma15-9||D4||12.70||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-10||205.9||5.83||Ma15-10||E4||14.17||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-11||97.6||12.30||Ma15-11||F4||7.70||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-12||129.5||9.27||Ma15-12||G4||10.73||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-13||263.1||4.56||Ma15-13||H4||15.44||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-14||76.1||15.77||Ma15-14||A5||4.23||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-15||138.7||8.65||Ma15-15||B5||11.35||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-16||216.4||5.55||Ma15-16||C5||14.45||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-17||137.6||8.72||Ma15-17||D5||11.28||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-18||224.6||5.34||Ma15-18||E5||14.66||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-19||111.7||10.74||Ma15-19||F5||9.26||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-20||289.9||4.14||Ma15-20||G5||15.86||1200.00||130.00||150.00 | |||
|- | |||
| Ma15-21||172.9||6.94||Ma15-21||H5||13.06||1200.00||130.00||150.00 | |||
|} | |||
{| {{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 1200ng''' | |||
| align="center" style="background:#f0f0f0;"|'''LabID ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Well IDs''' | |||
| 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)''' | |||
|- | |||
| SR-1||62.8||19.11||SR-1||A6||0.89||1200.00||130.00||150.00 | |||
|- | |||
| SR-2||90.8||13.22||SR-2||B6||6.78||1200.00||130.00||150.00 | |||
|- | |||
| SR-3||93.7||12.81||SR-3||C6||7.19||1200.00||130.00||150.00 | |||
|- | |||
| SR-4||98.0||12.24||SR-4||D6||7.76||1200.00||130.00||150.00 | |||
|- | |||
| SR-5||84.8||14.15||SR-5||E6||5.85||1200.00||130.00||150.00 | |||
|- | |||
| SR-6||78.6||15.27||SR-6||F6||4.73||1200.00||130.00||150.00 | |||
|- | |||
| SR-7||96.9||12.38||SR-7||G6||7.62||1200.00||130.00||150.00 | |||
|- | |||
| SR-8||91.9||13.06||SR-8||H6||6.94||1200.00||130.00||150.00 | |||
|- | |||
| SR-9||80.6||14.89||SR-9||A7||5.11||1200.00||130.00||150.00 | |||
|- | |||
| SR-10||184.8||6.49||SR-10||B7||13.51||1200.00||130.00||150.00 | |||
|- | |||
| SR-11||105.4||5.00||SR-11||C7||15.00||527.00||130.00||150.00 | |||
|- | |||
| SR-12||181.4||6.62||SR-12||D7||13.38||1200.00||130.00||150.00 | |||
|- | |||
| SR-13||270.0||4.44||SR-13||E7||15.56||1200.00||130.00||150.00 | |||
|- | |||
| SR-14||33.0||20.00||SR-14||F7||0.00||495.00||130.00||150.00 | |||
|- | |||
| SR-15||67.0||17.91||SR-15||G7||2.09||1200.00||130.00||150.00 | |||
|- | |||
| SR-16||145.6||8.24||SR-16||H7||11.76||1200.00||130.00||150.00 | |||
|- | |||
| SR-17||245.3||4.89||SR-17||A8||15.11||1200.00||130.00||150.00 | |||
|- | |||
| SR-18||89.9||13.35||SR-18||B8||6.65||1200.00||130.00||150.00 | |||
|- | |||
| SR-19||159.5||7.52||SR-19||C8||12.48||1200.00||130.00||150.00 | |||
|- | |||
| SR-20||195.7||6.13||SR-20||D8||13.87||1200.00||130.00||150.00 | |||
|- | |||
| SR-21||19.1||5.00||SR-21||E8||15.00||95.50||130.00||150.00 | |||
|- | |||
| SR-22||14.0||5.00||SR-22||F8||15.00||70.00||130.00||150.00 | |||
|} | |||
'''Program''' | |||
98°C for 10 minutes | |||
64°C for 2.5 hours | |||
4°C storage up to 20 hours | |||
Measure conc. with ssDNA Qubit assay (used 1ul of bis-cvt gDNA) | |||
== Qubit ssDNA assay == | |||
* Sample in assay 1ul, dilution 200x | |||
{| {{table}} border =1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''LabID ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Well IDs''' | |||
| align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Yields in 16ul (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''LabID ID''' | |||
| align="center" style="background:#f0f0f0;"|'''Well IDs''' | |||
| align="center" style="background:#f0f0f0;"|'''Repeated samples''' | |||
|- | |||
| 1.00||Ap15-1||A1||57.00||ng/mL||11.40||182.40||Ap15-1||A1||X | |||
|- | |||
| 2.00||Ap15-2||B1||62.20||ng/mL||12.44||199.04||Ap15-2||B1||X | |||
|- | |||
| 3.00||Ap15-3||C1||81.70||ng/mL||16.34||261.44||Ap15-3||C1||X | |||
|- | |||
| 4.00||Ap15-4||D1||75.40||ng/mL||15.08||241.28||Ap15-4||D1||X | |||
|- | |||
| 5.00||Ap15-5||E1||59.60||ng/mL||11.92||190.72||Ap15-5||E1||X | |||
|- | |||
| 6.00||Ap15-6||F1||74.50||ng/mL||14.90||238.40||Ap15-6||F1||X | |||
|- | |||
| 7.00||Ap15-7||G1||76.40||ng/mL||15.28||244.48||Ap15-7||G1||X | |||
|- | |||
| 8.00||Ap15-8||H1||76.40||ng/mL||15.28||244.48||Ap15-8||H1||X | |||
|- | |||
| 9.00||Ap15-9||A2||76.10||ng/mL||15.22||243.52||Ap15-9||A2||X | |||
|- | |||
| 10.00||Ap15-10||B2||60.10||ng/mL||12.02||192.32||Ap15-10||B2||X | |||
|- | |||
| 11.00||Ap15-11||C2||56.40||ng/mL||11.28||180.48||Ap15-11||C2||X | |||
|- | |||
| 12.00||Ap15-12||D2||59.00||ng/mL||11.80||188.80||Ap15-12||D2||X | |||
|- | |||
| 13.00||Ap15-13||E2||72.50||ng/mL||14.50||232.00||Ap15-13||E2||X | |||
|- | |||
| 14.00||Ap15-14||F2||74.30||ng/mL||14.86||237.76||Ap15-14||F2||X | |||
|- | |||
| 15.00||Ap15-15||G2||58.90||ng/mL||11.78||188.48||Ap15-15||G2||X | |||
|- | |||
| 16.00||Ap15-16||H2||45.10||ng/mL||9.02||144.32||Ap15-16||H2||X | |||
|- | |||
| 17.00||Ap15-17||A3||76.40||ng/mL||15.28||244.48||Ap15-17||A3||X | |||
|- | |||
| 18.00||Ap15-18||B3||54.90||ng/mL||10.98||175.68||Ap15-18||B3||X | |||
|- | |||
| 19.00||Ap15-19||C3||78.90||ng/mL||15.78||252.48||Ap15-19||C3||X | |||
|- | |||
| 20.00||Ma15-1||D3||44.10||ng/mL||8.82||141.12||Ma15-1||D3||X | |||
|- | |||
| 21.00||Ma15-2||E3||49.80||ng/mL||9.96||159.36||Ma15-2||E3||X | |||
|- | |||
| 22.00||Ma15-3||F3||51.60||ng/mL||10.32||165.12||Ma15-3||F3||X | |||
|- | |||
| 23.00||Ma15-4||G3||58.20||ng/mL||11.64||186.24||Ma15-4||G3||X | |||
|- | |||
| 24.00||Ma15-5||H3||60.10||ng/mL||12.02||192.32||Ma15-5||H3||X | |||
|- | |||
| 25.00||Ma15-6||A4||40.60||ng/mL||8.12||129.92||Ma15-6||A4||X | |||
|- | |||
| 26.00||Ma15-7||B4||59.90||ng/mL||11.98||191.68||Ma15-7||B4||X | |||
|- | |||
| 27.00||Ma15-8||C4||54.50||ng/mL||10.90||174.40||Ma15-8||C4||X | |||
|- | |||
| 28.00||Ma15-9||D4||43.60||ng/mL||8.72||139.52||Ma15-9||D4||X | |||
|- | |||
| 29.00||Ma15-10||E4||73.10||ng/mL||14.62||233.92||Ma15-10||E4||X | |||
|- | |||
| 30.00||Ma15-11||F4||49.70||ng/mL||9.94||159.04||Ma15-11||F4||X | |||
|- | |||
| 31.00||Ma15-12||G4||36.90||ng/mL||7.38||118.08||Ma15-12||G4||X | |||
|- | |||
| 32.00||Ma15-13||H4||60.00||ng/mL||12.00||192.00||Ma15-13||H4||X | |||
|- | |||
| 33.00||Ma15-14||A5||28.00||ng/mL||5.60||89.60||Ma15-14||A5||X | |||
|- | |||
| 34.00||Ma15-15||B5||27.70||ng/mL||5.54||88.64||Ma15-15||B5||X | |||
|- | |||
| 35.00||Ma15-16||C5||53.50||ng/mL||10.70||171.20||Ma15-16||C5||X | |||
|- | |||
| 36.00||Ma15-17||D5||51.10||ng/mL||10.22||163.52||Ma15-17||D5||X | |||
|- | |||
| 37.00||Ma15-18||E5||53.60||ng/mL||10.72||171.52||Ma15-18||E5||X | |||
|- | |||
| 38.00||Ma15-19||F5||51.40||ng/mL||10.28||164.48||Ma15-19||F5||X | |||
|- | |||
| 39.00||Ma15-20||G5||48.90||ng/mL||9.78||156.48||Ma15-20||G5||X | |||
|- | |||
| 40.00||Ma15-21||H5||55.40||ng/mL||11.08||177.28||Ma15-21||H5||X | |||
|- | |||
| 1.00||H9 p43 mTeSR||SR-1||108.00||ng/mL||21.60||345.60||SR-1||A6||- | |||
|- | |||
| 2.00||H9 p43 ACTIVIN ||SR-2||64.90||ng/mL||12.98||207.68||SR-2||B6||X | |||
|- | |||
| 3.00||H9 p43 BMP4||SR-3||133.00||ng/mL||26.60||425.60||SR-3||C6||- | |||
|- | |||
| 4.00||HUV0iPS4F1 p19||SR-4||140.00||ng/mL||28.00||448.00||SR-4||D6||- | |||
|- | |||
| 5.00||HUV-hiPS4F1 p20 ACTIVIN||SR-5||95.40||ng/mL||19.08||305.28||SR-5||E6||- | |||
|- | |||
| 6.00||HUV-hiPS4F1 p20 BMP4||SR-6||135.00||ng/mL||27.00||432.00||SR-6||F6||- | |||
|- | |||
| 7.00||HUV-iPS4F3 F19||SR-7||134.00||ng/mL||26.80||428.80||SR-7||G6||- | |||
|- | |||
| 8.00||HUV-hiPS4F3 p20 ACTIVIN||SR-8||96.10||ng/mL||19.22||307.52||SR-8||H6||- | |||
|- | |||
| 9.00||HUV-hiPS4F3 p20 BMP4||SR-9||120.00||ng/mL||24.00||384.00||SR-9||A7||- | |||
|- | |||
| 10.00||aMSCs p7||SR-10||53.90||ng/mL||10.78||172.48||SR-10||B7||X | |||
|- | |||
| 11.00||MSCiPS #8 p14 (~4 ul)||SR-11||71.60||ng/mL||14.32||229.12||SR-11||C7||Can\'t repeat | |||
|- | |||
| 12.00||MSCiPS #4 p14||SR-12||94.60||ng/mL||18.92||302.72||SR-12||D7||- | |||
|- | |||
| 13.00||hNiPS2F p23||SR-13||29.60||ng/mL||5.92||94.72||SR-13||E7||X | |||
|- | |||
| 14.00||hKiPS3F-7 p24 (~15ul)||SR-14||7.13||ng/mL||1.43||22.82||SR-14||F7||X | |||
|- | |||
| 15.00||hNSC ||SR-15||70.90||ng/mL||14.18||226.88||SR-15||G7||X | |||
|- | |||
| 16.00||hFiPS4F-7 p9||SR-16||23.10||ng/mL||4.62||73.92||SR-16||H7||X | |||
|- | |||
| 17.00||HUVEC p2||SR-17||50.80||ng/mL||10.16||162.56||SR-17||A8||X | |||
|- | |||
| 18.00||hiPS AST4F-4 p9||SR-18||131.00||ng/mL||26.20||419.20||SR-18||B8|| | |||
|- | |||
| 19.00||KERAT MMTA ||SR-19||19.80||ng/mL||3.96||63.36||SR-19||C8||X | |||
|- | |||
| 20.00||KiPS4F #8||SR-20||19.00||ng/mL||3.80||60.80||SR-20||D8||X | |||
|- | |||
| 21.00||olf MSC 19.1ng/ul volume only 3-5ul||SR-21||4.60||ng/mL||0.92||14.72||SR-21||E8||can\'t repeat | |||
|- | |||
| 22.00||KiPS 14ng/ul volume only 3-5ul||SR-22||7.42||ng/mL||1.48||23.74||SR-22||F8||can\'t repeat | |||
|} | |||
* From the yields of bis-cvt gDNA, many samples have the yields lower than 300ng or 200ng which is the amount the acceptable for capture with BSPP. I checked the yields of the old samples I have processed before (SR-1 - SR-9) to see if this is the technical error of this batch. I got the total yields of SR-1 -15 pretty much the same as I got before. This suggested that there are not the batch effect, but may be the quality of gDNA. I need to repeat bis-cvt again to get enough yield for capture. However, there are some sample that has no more gDNA or has very low concentration, and I don't expected to get higher yield from those samples. | |||
* Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-21 |
Latest revision as of 16:03, 26 August 2012
DMR 330k BSPP capture of samples from Sergio[edit]
Sample list[edit]
New gDNA extracted from the cell pellet[edit]
on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-5-17
No. | Sample ID | Lab ID | Conc. (ng/ul) | Volume (ul) | Yields (ug) |
1 | HUES2 p41 | Ap15-1 | 110.0 | 250 | 27.5 |
2 | ASThiPS4F1 p12 | Ap15-2 | 139.1 | 250 | 34.8 |
3 | ASThiPS4F2 p12 | Ap15-3 | 145.1 | 250 | 36.3 |
4 | HUES6 p39 Control TeSR | Ap15-4 | 295.5 | 250 | 73.9 |
5 | HUES6 p39 ACTIVIN | Ap15-5 | 128.1 | 250 | 32.0 |
6 | HUES6 p39 BMP-4 | Ap15-6 | 153.9 | 250 | 38.5 |
7 | HUES9 p39 Control TeSR | Ap15-7 | 183.5 | 250 | 45.9 |
8 | HUES9 p39 ACTIVIN | Ap15-8 | 149.3 | 250 | 37.3 |
9 | HUES9 p39 BMP-4 | Ap15-9 | 111.2 | 250 | 27.8 |
10 | HUES3 p36 Control TeSR | Ap15-10 | 153.5 | 250 | 38.4 |
11 | HUES3 p36 ACTIVIN | Ap15-11 | 87.5 | 250 | 21.9 |
12 | HUES3 p36 BMP-4 | Ap15-12 | 109.7 | 250 | 27.4 |
13 | H1 p54 Control TeSR | Ap15-13 | 191.3 | 250 | 47.8 |
14 | H1 p54 ACTIVIN | Ap15-14 | 108.7 | 250 | 27.2 |
15 | ASThiPS4F5 p16 Control TeSR | Ap15-15 | 134.2 | 250 | 33.6 |
*16 | ASThiPS4F5 p16 ACTIVIN | Ap15-16 | 50.2 | 500 | 25.1 |
17 | ASThiPS4F4 p36 Control TeSR | Ap15-17 | 183.0 | 250 | 45.8 |
18 | ASThiPS4F4 p36 ACTIVIN | Ap15-18 | 82.6 | 250 | 20.7 |
19 | ASThiPS4F4 p36 BMP-4 | Ap15-19 | 263.4 | 250 | 65.9 |
20 | H1 p48 | Ma15-2 | 215.2 | 200 | 43.0 |
21 | H9 p47 | Ma15-3 | 252.0 | 200 | 50.4 |
22 | HUES3 p33 | Ma15-4 | 226.1 | 200 | 45.2 |
23 | HUES6 p26 | Ma15-5 | 238.7 | 200 | 47.7 |
24 | HUES8 p35 | Ma15-6 | 83.4 | 200 | 16.7 |
25 | HUES9 p36 | Ma15-7 | 236.8 | 200 | 47.4 |
26 | ASThiPS4F3 p24 (derived from Astrocytes) | Ma15-8 | 175.9 | 200 | 35.2 |
27 | ASThiPS4F4 p16 (derived from Astrocytes) | Ma15-9 | 164.3 | 200 | 32.9 |
28 | FiPS4F2 p32 (derived from IMR90 fibroblasts) | Ma15-10 | 205.9 | 200 | 41.2 |
29 | FiPS4F5 p29 (derived from IMR90 fibroblasts) | Ma15-11 | 97.6 | 200 | 19.5 |
30 | KiPS4FA p54 (derived from keratinocytes) | Ma15-12 | 129.5 | 200 | 25.9 |
31 | Huv-iPS4F6 p23 (derived from HUVECs) | Ma15-13 | 263.1 | 200 | 52.6 |
32 | IMR90 p6 | Ma15-14 | 76.1 | 200 | 15.2 |
33 | Keratinocytes p3 | Ma15-15 | 138.7 | 200 | 27.7 |
34 | H1 p47 Control | Ma15-16 | 216.4 | 200 | 43.3 |
35 | H1 p47 BMP-4 | Ma15-17 | 137.6 | 200 | 27.5 |
36 | ASThiPS4F5 p12 Control | Ma15-18 | 224.6 | 200 | 44.9 |
37 | ASThiPS4F5 p12 BMP-4 | Ma15-19 | 111.7 | 200 | 22.3 |
38 | Huv-iPS4F6 p23 Control | Ma15-20 | 289.9 | 200 | 58.0 |
39 | Huv-iPS4F6 p23 BMP-4 | Ma15-21 | 172.9 | 200 | 34.6 |
40 | Huv-iPS4F6 p23 ACTIVIN | Ma15-1 | 86.7 | 250 | 21.7 |
Old gDNA of samples have been captured in the previous experiments[edit]
- Note:
- Some of gDNA concentrations were re-measured and note that some of them have conc. lower than 50ng/ul, and some of them don't have enough amount for bisulfite conversion
- Samples KERAT MMTA and KiPS4F #8 have high conc., but very little volume. I added ~17ul H2O then re-measured conc. again
- ***I may not do bis-cvt on the two samples, SR-21 and SR22 since the amount of gDNA was too little
No | Sample IDs | Lab IDs | Conc. (ng/ul) |
1 | H9 p43 mTeSR | SR-1 | 62.8 |
2 | H9 p43 ACTIVIN | SR-2 | 90.8 |
3 | H9 p43 BMP4 | SR-3 | 93.7 |
4 | HUV0iPS4F1 p19 | SR-4 | 98.0 |
5 | HUV-hiPS4F1 p20 ACTIVIN | SR-5 | 84.8 |
6 | HUV-hiPS4F1 p20 BMP4 | SR-6 | 78.6 |
7 | HUV-iPS4F3 F19 | SR-7 | 96.9 |
8 | HUV-hiPS4F3 p20 ACTIVIN | SR-8 | 91.9 |
9 | HUV-hiPS4F3 p20 BMP4 | SR-9 | 80.6 |
10 | aMSCs p7 | SR-10 | 184.8 |
11 | MSCiPS #8 p14 (~4 ul) | SR-11 | 105.4 |
12 | MSCiPS #4 p14 | SR-12 | 181.4 |
13 | hNiPS2F p23 | SR-13 | 270.0 |
14 | hKiPS3F-7 p24 (~15ul) | SR-14 | 33.0 |
15 | hNSC | SR-15 | 67.0 |
16 | hFiPS4F-7 p9 | SR-16 | 145.6 |
17 | HUVEC p2 | SR-17 | 245.3 |
18 | hiPS AST4F-4 p9 | SR-18 | 89.9 |
19 | KERAT MMTA | SR-19 | 159.5 |
20 | KiPS4F #8 | SR-20 | 195.7 |
21 | ***olf MSC 19.1ng/ul volume only 3-5ul | SR-21 | 19.1 |
22 | ***KiPS 14ng/ul volume only 3-5ul | SR-22 | 14.0 |
8 samples of bis-cvt gDNA from Dinh[edit]
- The list will be filled later and need to re-measure of the conc. with ssDNA Qubit assay
Bisulfite conversion[edit]
Reagent prep Add the following reagents to CT Conversion Reagent - H2O 9.0mL - M-Dissolving Buffer 500ul - M-Dilution Buffer 3.0mL Mix at room temperature with frequent vortexing or shaking for 15 minutes Add 144 ml of 100% ethanol to the 36 ml M-Wash Buffer concentrate before use
Lab IDs | Conc. (ng/ul) | Volume for 1200ng | LabID ID | Well IDs | H2O | Total amount (ng) | CT conversion reagent (ul) | Total volume (ul) |
Ap15-1 | 110.0 | 10.91 | Ap15-1 | A1 | 9.09 | 1200.00 | 130.00 | 150.00 |
Ap15-2 | 139.1 | 8.63 | Ap15-2 | B1 | 11.37 | 1200.00 | 130.00 | 150.00 |
Ap15-3 | 145.1 | 8.27 | Ap15-3 | C1 | 11.73 | 1200.00 | 130.00 | 150.00 |
Ap15-4 | 295.5 | 4.06 | Ap15-4 | D1 | 15.94 | 1200.00 | 130.00 | 150.00 |
Ap15-5 | 128.1 | 9.37 | Ap15-5 | E1 | 10.63 | 1200.00 | 130.00 | 150.00 |
Ap15-6 | 153.9 | 7.80 | Ap15-6 | F1 | 12.20 | 1200.00 | 130.00 | 150.00 |
Ap15-7 | 183.5 | 6.54 | Ap15-7 | G1 | 13.46 | 1200.00 | 130.00 | 150.00 |
Ap15-8 | 149.3 | 8.04 | Ap15-8 | H1 | 11.96 | 1200.00 | 130.00 | 150.00 |
Ap15-9 | 111.2 | 10.79 | Ap15-9 | A2 | 9.21 | 1200.00 | 130.00 | 150.00 |
Ap15-10 | 153.5 | 7.82 | Ap15-10 | B2 | 12.18 | 1200.00 | 130.00 | 150.00 |
Ap15-11 | 87.5 | 13.71 | Ap15-11 | C2 | 6.29 | 1200.00 | 130.00 | 150.00 |
Ap15-12 | 109.7 | 10.94 | Ap15-12 | D2 | 9.06 | 1200.00 | 130.00 | 150.00 |
Ap15-13 | 191.3 | 6.27 | Ap15-13 | E2 | 13.73 | 1200.00 | 130.00 | 150.00 |
Ap15-14 | 108.7 | 11.04 | Ap15-14 | F2 | 8.96 | 1200.00 | 130.00 | 150.00 |
Ap15-15 | 134.2 | 8.94 | Ap15-15 | G2 | 11.06 | 1200.00 | 130.00 | 150.00 |
Ap15-16 | 50.2 | 20.00 | Ap15-16 | H2 | 0.00 | 1200.00 | 130.00 | 150.00 |
Ap15-17 | 183.0 | 6.56 | Ap15-17 | A3 | 13.44 | 1200.00 | 130.00 | 150.00 |
Ap15-18 | 82.6 | 14.53 | Ap15-18 | B3 | 5.47 | 1200.00 | 130.00 | 150.00 |
Ap15-19 | 263.4 | 4.56 | Ap15-19 | C3 | 15.44 | 1200.00 | 130.00 | 150.00 |
Ma15-1 | 86.7 | 13.84 | Ma15-1 | D3 | 6.16 | 1200.00 | 130.00 | 150.00 |
Ma15-2 | 215.2 | 5.58 | Ma15-2 | E3 | 14.42 | 1200.00 | 130.00 | 150.00 |
Ma15-3 | 252.0 | 4.76 | Ma15-3 | F3 | 15.24 | 1200.00 | 130.00 | 150.00 |
Ma15-4 | 226.1 | 5.31 | Ma15-4 | G3 | 14.69 | 1200.00 | 130.00 | 150.00 |
Ma15-5 | 238.7 | 5.03 | Ma15-5 | H3 | 14.97 | 1200.00 | 130.00 | 150.00 |
Ma15-6 | 83.4 | 14.39 | Ma15-6 | A4 | 5.61 | 1200.00 | 130.00 | 150.00 |
Ma15-7 | 236.8 | 5.07 | Ma15-7 | B4 | 14.93 | 1200.00 | 130.00 | 150.00 |
Ma15-8 | 175.9 | 6.82 | Ma15-8 | C4 | 13.18 | 1200.00 | 130.00 | 150.00 |
Ma15-9 | 164.3 | 7.30 | Ma15-9 | D4 | 12.70 | 1200.00 | 130.00 | 150.00 |
Ma15-10 | 205.9 | 5.83 | Ma15-10 | E4 | 14.17 | 1200.00 | 130.00 | 150.00 |
Ma15-11 | 97.6 | 12.30 | Ma15-11 | F4 | 7.70 | 1200.00 | 130.00 | 150.00 |
Ma15-12 | 129.5 | 9.27 | Ma15-12 | G4 | 10.73 | 1200.00 | 130.00 | 150.00 |
Ma15-13 | 263.1 | 4.56 | Ma15-13 | H4 | 15.44 | 1200.00 | 130.00 | 150.00 |
Ma15-14 | 76.1 | 15.77 | Ma15-14 | A5 | 4.23 | 1200.00 | 130.00 | 150.00 |
Ma15-15 | 138.7 | 8.65 | Ma15-15 | B5 | 11.35 | 1200.00 | 130.00 | 150.00 |
Ma15-16 | 216.4 | 5.55 | Ma15-16 | C5 | 14.45 | 1200.00 | 130.00 | 150.00 |
Ma15-17 | 137.6 | 8.72 | Ma15-17 | D5 | 11.28 | 1200.00 | 130.00 | 150.00 |
Ma15-18 | 224.6 | 5.34 | Ma15-18 | E5 | 14.66 | 1200.00 | 130.00 | 150.00 |
Ma15-19 | 111.7 | 10.74 | Ma15-19 | F5 | 9.26 | 1200.00 | 130.00 | 150.00 |
Ma15-20 | 289.9 | 4.14 | Ma15-20 | G5 | 15.86 | 1200.00 | 130.00 | 150.00 |
Ma15-21 | 172.9 | 6.94 | Ma15-21 | H5 | 13.06 | 1200.00 | 130.00 | 150.00 |
Lab IDs | Conc. (ng/ul) | Volume for 1200ng | LabID ID | Well IDs | H2O | Total amount (ng) | CT conversion reagent (ul) | Total volume (ul) |
SR-1 | 62.8 | 19.11 | SR-1 | A6 | 0.89 | 1200.00 | 130.00 | 150.00 |
SR-2 | 90.8 | 13.22 | SR-2 | B6 | 6.78 | 1200.00 | 130.00 | 150.00 |
SR-3 | 93.7 | 12.81 | SR-3 | C6 | 7.19 | 1200.00 | 130.00 | 150.00 |
SR-4 | 98.0 | 12.24 | SR-4 | D6 | 7.76 | 1200.00 | 130.00 | 150.00 |
SR-5 | 84.8 | 14.15 | SR-5 | E6 | 5.85 | 1200.00 | 130.00 | 150.00 |
SR-6 | 78.6 | 15.27 | SR-6 | F6 | 4.73 | 1200.00 | 130.00 | 150.00 |
SR-7 | 96.9 | 12.38 | SR-7 | G6 | 7.62 | 1200.00 | 130.00 | 150.00 |
SR-8 | 91.9 | 13.06 | SR-8 | H6 | 6.94 | 1200.00 | 130.00 | 150.00 |
SR-9 | 80.6 | 14.89 | SR-9 | A7 | 5.11 | 1200.00 | 130.00 | 150.00 |
SR-10 | 184.8 | 6.49 | SR-10 | B7 | 13.51 | 1200.00 | 130.00 | 150.00 |
SR-11 | 105.4 | 5.00 | SR-11 | C7 | 15.00 | 527.00 | 130.00 | 150.00 |
SR-12 | 181.4 | 6.62 | SR-12 | D7 | 13.38 | 1200.00 | 130.00 | 150.00 |
SR-13 | 270.0 | 4.44 | SR-13 | E7 | 15.56 | 1200.00 | 130.00 | 150.00 |
SR-14 | 33.0 | 20.00 | SR-14 | F7 | 0.00 | 495.00 | 130.00 | 150.00 |
SR-15 | 67.0 | 17.91 | SR-15 | G7 | 2.09 | 1200.00 | 130.00 | 150.00 |
SR-16 | 145.6 | 8.24 | SR-16 | H7 | 11.76 | 1200.00 | 130.00 | 150.00 |
SR-17 | 245.3 | 4.89 | SR-17 | A8 | 15.11 | 1200.00 | 130.00 | 150.00 |
SR-18 | 89.9 | 13.35 | SR-18 | B8 | 6.65 | 1200.00 | 130.00 | 150.00 |
SR-19 | 159.5 | 7.52 | SR-19 | C8 | 12.48 | 1200.00 | 130.00 | 150.00 |
SR-20 | 195.7 | 6.13 | SR-20 | D8 | 13.87 | 1200.00 | 130.00 | 150.00 |
SR-21 | 19.1 | 5.00 | SR-21 | E8 | 15.00 | 95.50 | 130.00 | 150.00 |
SR-22 | 14.0 | 5.00 | SR-22 | F8 | 15.00 | 70.00 | 130.00 | 150.00 |
Program 98°C for 10 minutes 64°C for 2.5 hours 4°C storage up to 20 hours Measure conc. with ssDNA Qubit assay (used 1ul of bis-cvt gDNA)
Qubit ssDNA assay[edit]
- Sample in assay 1ul, dilution 200x
Sample | LabID ID | Well IDs | Concentration in the Qubit | ' | Final conc. (ng/ul) | Yields in 16ul (ng) | LabID ID | Well IDs | Repeated samples |
1.00 | Ap15-1 | A1 | 57.00 | ng/mL | 11.40 | 182.40 | Ap15-1 | A1 | X |
2.00 | Ap15-2 | B1 | 62.20 | ng/mL | 12.44 | 199.04 | Ap15-2 | B1 | X |
3.00 | Ap15-3 | C1 | 81.70 | ng/mL | 16.34 | 261.44 | Ap15-3 | C1 | X |
4.00 | Ap15-4 | D1 | 75.40 | ng/mL | 15.08 | 241.28 | Ap15-4 | D1 | X |
5.00 | Ap15-5 | E1 | 59.60 | ng/mL | 11.92 | 190.72 | Ap15-5 | E1 | X |
6.00 | Ap15-6 | F1 | 74.50 | ng/mL | 14.90 | 238.40 | Ap15-6 | F1 | X |
7.00 | Ap15-7 | G1 | 76.40 | ng/mL | 15.28 | 244.48 | Ap15-7 | G1 | X |
8.00 | Ap15-8 | H1 | 76.40 | ng/mL | 15.28 | 244.48 | Ap15-8 | H1 | X |
9.00 | Ap15-9 | A2 | 76.10 | ng/mL | 15.22 | 243.52 | Ap15-9 | A2 | X |
10.00 | Ap15-10 | B2 | 60.10 | ng/mL | 12.02 | 192.32 | Ap15-10 | B2 | X |
11.00 | Ap15-11 | C2 | 56.40 | ng/mL | 11.28 | 180.48 | Ap15-11 | C2 | X |
12.00 | Ap15-12 | D2 | 59.00 | ng/mL | 11.80 | 188.80 | Ap15-12 | D2 | X |
13.00 | Ap15-13 | E2 | 72.50 | ng/mL | 14.50 | 232.00 | Ap15-13 | E2 | X |
14.00 | Ap15-14 | F2 | 74.30 | ng/mL | 14.86 | 237.76 | Ap15-14 | F2 | X |
15.00 | Ap15-15 | G2 | 58.90 | ng/mL | 11.78 | 188.48 | Ap15-15 | G2 | X |
16.00 | Ap15-16 | H2 | 45.10 | ng/mL | 9.02 | 144.32 | Ap15-16 | H2 | X |
17.00 | Ap15-17 | A3 | 76.40 | ng/mL | 15.28 | 244.48 | Ap15-17 | A3 | X |
18.00 | Ap15-18 | B3 | 54.90 | ng/mL | 10.98 | 175.68 | Ap15-18 | B3 | X |
19.00 | Ap15-19 | C3 | 78.90 | ng/mL | 15.78 | 252.48 | Ap15-19 | C3 | X |
20.00 | Ma15-1 | D3 | 44.10 | ng/mL | 8.82 | 141.12 | Ma15-1 | D3 | X |
21.00 | Ma15-2 | E3 | 49.80 | ng/mL | 9.96 | 159.36 | Ma15-2 | E3 | X |
22.00 | Ma15-3 | F3 | 51.60 | ng/mL | 10.32 | 165.12 | Ma15-3 | F3 | X |
23.00 | Ma15-4 | G3 | 58.20 | ng/mL | 11.64 | 186.24 | Ma15-4 | G3 | X |
24.00 | Ma15-5 | H3 | 60.10 | ng/mL | 12.02 | 192.32 | Ma15-5 | H3 | X |
25.00 | Ma15-6 | A4 | 40.60 | ng/mL | 8.12 | 129.92 | Ma15-6 | A4 | X |
26.00 | Ma15-7 | B4 | 59.90 | ng/mL | 11.98 | 191.68 | Ma15-7 | B4 | X |
27.00 | Ma15-8 | C4 | 54.50 | ng/mL | 10.90 | 174.40 | Ma15-8 | C4 | X |
28.00 | Ma15-9 | D4 | 43.60 | ng/mL | 8.72 | 139.52 | Ma15-9 | D4 | X |
29.00 | Ma15-10 | E4 | 73.10 | ng/mL | 14.62 | 233.92 | Ma15-10 | E4 | X |
30.00 | Ma15-11 | F4 | 49.70 | ng/mL | 9.94 | 159.04 | Ma15-11 | F4 | X |
31.00 | Ma15-12 | G4 | 36.90 | ng/mL | 7.38 | 118.08 | Ma15-12 | G4 | X |
32.00 | Ma15-13 | H4 | 60.00 | ng/mL | 12.00 | 192.00 | Ma15-13 | H4 | X |
33.00 | Ma15-14 | A5 | 28.00 | ng/mL | 5.60 | 89.60 | Ma15-14 | A5 | X |
34.00 | Ma15-15 | B5 | 27.70 | ng/mL | 5.54 | 88.64 | Ma15-15 | B5 | X |
35.00 | Ma15-16 | C5 | 53.50 | ng/mL | 10.70 | 171.20 | Ma15-16 | C5 | X |
36.00 | Ma15-17 | D5 | 51.10 | ng/mL | 10.22 | 163.52 | Ma15-17 | D5 | X |
37.00 | Ma15-18 | E5 | 53.60 | ng/mL | 10.72 | 171.52 | Ma15-18 | E5 | X |
38.00 | Ma15-19 | F5 | 51.40 | ng/mL | 10.28 | 164.48 | Ma15-19 | F5 | X |
39.00 | Ma15-20 | G5 | 48.90 | ng/mL | 9.78 | 156.48 | Ma15-20 | G5 | X |
40.00 | Ma15-21 | H5 | 55.40 | ng/mL | 11.08 | 177.28 | Ma15-21 | H5 | X |
1.00 | H9 p43 mTeSR | SR-1 | 108.00 | ng/mL | 21.60 | 345.60 | SR-1 | A6 | - |
2.00 | H9 p43 ACTIVIN | SR-2 | 64.90 | ng/mL | 12.98 | 207.68 | SR-2 | B6 | X |
3.00 | H9 p43 BMP4 | SR-3 | 133.00 | ng/mL | 26.60 | 425.60 | SR-3 | C6 | - |
4.00 | HUV0iPS4F1 p19 | SR-4 | 140.00 | ng/mL | 28.00 | 448.00 | SR-4 | D6 | - |
5.00 | HUV-hiPS4F1 p20 ACTIVIN | SR-5 | 95.40 | ng/mL | 19.08 | 305.28 | SR-5 | E6 | - |
6.00 | HUV-hiPS4F1 p20 BMP4 | SR-6 | 135.00 | ng/mL | 27.00 | 432.00 | SR-6 | F6 | - |
7.00 | HUV-iPS4F3 F19 | SR-7 | 134.00 | ng/mL | 26.80 | 428.80 | SR-7 | G6 | - |
8.00 | HUV-hiPS4F3 p20 ACTIVIN | SR-8 | 96.10 | ng/mL | 19.22 | 307.52 | SR-8 | H6 | - |
9.00 | HUV-hiPS4F3 p20 BMP4 | SR-9 | 120.00 | ng/mL | 24.00 | 384.00 | SR-9 | A7 | - |
10.00 | aMSCs p7 | SR-10 | 53.90 | ng/mL | 10.78 | 172.48 | SR-10 | B7 | X |
11.00 | MSCiPS #8 p14 (~4 ul) | SR-11 | 71.60 | ng/mL | 14.32 | 229.12 | SR-11 | C7 | Can\'t repeat |
12.00 | MSCiPS #4 p14 | SR-12 | 94.60 | ng/mL | 18.92 | 302.72 | SR-12 | D7 | - |
13.00 | hNiPS2F p23 | SR-13 | 29.60 | ng/mL | 5.92 | 94.72 | SR-13 | E7 | X |
14.00 | hKiPS3F-7 p24 (~15ul) | SR-14 | 7.13 | ng/mL | 1.43 | 22.82 | SR-14 | F7 | X |
15.00 | hNSC | SR-15 | 70.90 | ng/mL | 14.18 | 226.88 | SR-15 | G7 | X |
16.00 | hFiPS4F-7 p9 | SR-16 | 23.10 | ng/mL | 4.62 | 73.92 | SR-16 | H7 | X |
17.00 | HUVEC p2 | SR-17 | 50.80 | ng/mL | 10.16 | 162.56 | SR-17 | A8 | X |
18.00 | hiPS AST4F-4 p9 | SR-18 | 131.00 | ng/mL | 26.20 | 419.20 | SR-18 | B8 | |
19.00 | KERAT MMTA | SR-19 | 19.80 | ng/mL | 3.96 | 63.36 | SR-19 | C8 | X |
20.00 | KiPS4F #8 | SR-20 | 19.00 | ng/mL | 3.80 | 60.80 | SR-20 | D8 | X |
21.00 | olf MSC 19.1ng/ul volume only 3-5ul | SR-21 | 4.60 | ng/mL | 0.92 | 14.72 | SR-21 | E8 | can\'t repeat |
22.00 | KiPS 14ng/ul volume only 3-5ul | SR-22 | 7.42 | ng/mL | 1.48 | 23.74 | SR-22 | F8 | can\'t repeat |
- From the yields of bis-cvt gDNA, many samples have the yields lower than 300ng or 200ng which is the amount the acceptable for capture with BSPP. I checked the yields of the old samples I have processed before (SR-1 - SR-9) to see if this is the technical error of this batch. I got the total yields of SR-1 -15 pretty much the same as I got before. This suggested that there are not the batch effect, but may be the quality of gDNA. I need to repeat bis-cvt again to get enough yield for capture. However, there are some sample that has no more gDNA or has very low concentration, and I don't expected to get higher yield from those samples.