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= Bisulfite Conversion = * Used EZ-96 DNA Methylation-Goldβ’ Kit (deep well format) Cat#D5008 2011_09_17<br> * Note: ** I just realized that once I got the the first batch Penn samples, I aliquoted DNA sample to 20ul (plate1) and 30 ul (plate2). I still have the samples I understand that I used up. ** I will used "plate" for the Penn samples stored in the lab and "tube" for the samples I just got from Tishkoff's lab ** I also got three additional samples from Alice (from Yang Xu's lab) including, Hues1, Hues3 and Hues8 in cell pellets. After extracted gDNA and eluted 150ul each for elute1 and elute2, the concentration was measure by Nanodrop. ''' Plate layout of bisulfite conversion''' {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Plate layout''' | align="center" style="background:#f0f0f0;"|'''1.00''' | align="center" style="background:#f0f0f0;"|'''2.00''' | align="center" style="background:#f0f0f0;"|'''3.00''' | align="center" style="background:#f0f0f0;"|'''4.00''' | align="center" style="background:#f0f0f0;"|'''5.00''' | align="center" style="background:#f0f0f0;"|'''6.00''' | align="center" style="background:#f0f0f0;"|'''7.00''' | align="center" style="background:#f0f0f0;"|'''8.00''' | align="center" style="background:#f0f0f0;"|'''9.00''' | align="center" style="background:#f0f0f0;"|'''10.00''' | align="center" style="background:#f0f0f0;"|'''11.00''' | align="center" style="background:#f0f0f0;"|'''12.00''' |- | A||Sergio1||||Sergio9||||GM12802 (wellID E7)||||NA12156 (wellID B3)-4||||218 HFF P7||||PGP1iPS|| |- | B||Sergio2||||GM11995 (wellID G7)||||GM12815 (wellID G12)||||NA12156 (wellID B3)-5||||KEBR028A (B4-plate2)-1||KEBR028A (B4-plate2)-2|||| |- | C||Sergio3||||GM11993 (wellID F2)||||GM12812 (wellID F11)||||NA12156 (wellID B3)-6||||KEBR028A (tube)-1||KEBR028A (tube)-2||Hues1||Hues1 |- | D||Sergio4||||GM10861 (wellID A9)||||GM12814 (wellID A7)||||NA12156 (wellID B3)-7||||KEBR040 (D4-plate1)||KEBR040 (D4-plate2)||Hues3||Hues3 |- | E||Sergio5||||GM11992 (wellID C2 )||||GM12801 (wellID D9)||||NA12156 (wellID B3)-8||||TZHZ018 (F1-plate1)||TZHZ018 (F1-plate2)||Hues8||Hues8 |- | F||Sergio6||||GM11994 (wellID G9 )||||NA12156 (wellID B3)-1||||NA12156 (wellID B3)-9||||TZHZ031A (tube)-1||TZHZ031A (tube)-2|||| |- | G||Sergio7||||GM10860 (wellID F10)||||NA12156 (wellID B3)-2||||NA12156 (wellID B3)-10||||TZHZ041A (tube)-1||TZHZ041A (tube)-2|||| |- | H||Sergio8||||GM12813 (wellID E8)||||NA12156 (wellID B3)-3||||2HFF P6||||TZHZ075 (H01-plate2)-1||TZHZ075 (H01-plate2)-1|||| |}<br> {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Order''' | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Family''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''Volume for 1000ng''' | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''H2O''' | align="center" style="background:#f0f0f0;"|'''Total amount (ng)''' | align="center" style="background:#f0f0f0;"|'''CT conversion reagent (ul)''' |- | 1||1. H9||||62.80||15.92||A1||1. H9||4.08||1000.00||130.00 |- | 2||2. H9+Activin 100ng/mL 5days||||90.80||11.01||B1||2. H9+Activin 100ng/mL 5days||8.99||1000.00||130.00 |- | 3||3. H9+BMP4 50ng/mL 5days||||93.70||10.67||C1||3. H9+BMP4 50ng/mL 5days||9.33||1000.00||130.00 |- | 4||4. HUViPS4F1||||98.00||10.20||D1||4. HUViPS4F1||9.80||1000.00||130.00 |- | 5||5. HUViPS4F1+Activin 100ng/mL 5days||||84.80||11.79||E1||5. HUViPS4F1+Activin 100ng/mL 5days||8.21||1000.00||130.00 |- | 6||6. HUViPS4F1+BMP4 50ng/mL 5days||||78.60||12.72||F1||6. HUViPS4F1+BMP4 50ng/mL 5days||7.28||1000.00||130.00 |- | 7||7. HUViPS4F3||||96.90||10.32||G1||7. HUViPS4F3||9.68||1000.00||130.00 |- | 8||8. HUViPS4F3+Activin 100ng/mL 5days||||91.90||10.88||H1||8. HUViPS4F3+Activin 100ng/mL 5days||9.12||1000.00||130.00 |- | 9||9. HUViPS4F3+BMP4 50ng/mL 5days||||80.60||12.41||A3||9. HUViPS4F3+BMP4 50ng/mL 5days||7.59||1000.00||130.00 |- | 10||GM11995 (wellID G7)||1362||200.00||5.00||B3||GM11995 (wellID G7)||15.00||1000.00||130.00 |- | 11||GM11993 (wellID F2)||1362||200.00||5.00||C3||GM11993 (wellID F2)||15.00||1000.00||130.00 |- | 12||GM10861 (wellID A9)||1362||200.00||5.00||D3||GM10861 (wellID A9)||15.00||1000.00||130.00 |- | 13||GM11992 (wellID C2 )||1362||200.00||5.00||E3||GM11992 (wellID C2 )||15.00||1000.00||130.00 |- | 14||GM11994 (wellID G9 )||1362||200.00||5.00||F3||GM11994 (wellID G9 )||15.00||1000.00||130.00 |- | 15||GM10860 (wellID F10)||1362||200.00||5.00||G3||GM10860 (wellID F10)||15.00||1000.00||130.00 |- | 16||GM12813 (wellID E8)||1454||200.00||5.00||H3||GM12813 (wellID E8)||15.00||1000.00||130.00 |- | 17||GM12802 (wellID E7)||1454||200.00||5.00||A5||GM12802 (wellID E7)||15.00||1000.00||130.00 |- | 18||GM12815 (wellID G12)||1454||200.00||5.00||B5||GM12815 (wellID G12)||15.00||1000.00||130.00 |- | 19||GM12812 (wellID F11)||1454||200.00||5.00||C5||GM12812 (wellID F11)||15.00||1000.00||130.00 |- | 20||GM12814 (wellID A7)||1454||200.00||5.00||D5||GM12814 (wellID A7)||15.00||1000.00||130.00 |- | 21||GM12801 (wellID D9)||1454||200.00||5.00||E5||GM12801 (wellID D9)||15.00||1000.00||130.00 |- | 22||NA12156 (wellID B3)-1||1408||200.00||5.00||F5||NA12156 (wellID B3)-1||15.00||1000.00||130.00 |- | 23||NA12156 (wellID B3)-2||1408||200.00||5.00||G5||NA12156 (wellID B3)-2||15.00||1000.00||130.00 |- | 24||NA12156 (wellID B3)-3||1408||200.00||5.00||H5||NA12156 (wellID B3)-3||15.00||1000.00||130.00 |- | 25||NA12156 (wellID B3)-4||1408||200.00||5.00||A7||NA12156 (wellID B3)-4||15.00||1000.00||130.00 |- | 26||NA12156 (wellID B3)-5||1408||200.00||5.00||B7||NA12156 (wellID B3)-5||15.00||1000.00||130.00 |- | 27||NA12156 (wellID B3)-6||1408||200.00||5.00||C7||NA12156 (wellID B3)-6||15.00||1000.00||130.00 |- | 28||NA12156 (wellID B3)-7||1408||200.00||5.00||D7||NA12156 (wellID B3)-7||15.00||1000.00||130.00 |- | 29||NA12156 (wellID B3)-8||1408||200.00||5.00||E7||NA12156 (wellID B3)-8||15.00||1000.00||130.00 |- | 30||NA12156 (wellID B3)-9||1408||200.00||5.00||F7||NA12156 (wellID B3)-9||15.00||1000.00||130.00 |- | 31||NA12156 (wellID B3)-10||1408||200.00||5.00||G7||NA12156 (wellID B3)-10||15.00||1000.00||130.00 |- | 32||2HFF P6||||280.10||3.57||H7||2HFF P6||16.43||1000.00||130.00 |- | 33||218 HFF P7||||446.10||2.24||A9||218 HFF P7||17.76||1000.00||130.00 |- | 34||KEBR028A (B4-plate2)-1,2||||170.70||5.86||B9,B10||KEBR028A (B4-plate2)-1||14.14||1000.00||130.00 |- | 35||KEBR028A (tube)-1,2||||23.80||20.00||C9,C10||KEBR028A (tube)-1||0.00||476.00||130.00 |- | 36||KEBR040 (D4-plate1)||||173.50||5.76||D9||KEBR040 (D4-plate1)||14.24||1000.00||130.00 |- | ||KEBR040 (D4-plate2)||||111.50||8.97||D10||KEBR040 (D4-plate2)||11.03||1000.00||130.00 |- | 37||TZHZ018 (F1-plate1)||||185.20||5.40||E9||TZHZ018 (F1-plate1)||14.60||1000.00||130.00 |- | ||TZHZ018 (F1-plate2)||||196.10||5.10||E10||TZHZ018 (F1-plate2)||14.90||1000.00||130.00 |- | 38||TZHZ031A (tube)-1,2||||55.10||18.15||F9,F10||TZHZ031A (tube)-1||1.85||1000.00||130.00 |- | 39||TZHZ041A (tube)-1,2||||54.60||18.32||G9,G10||TZHZ041A (tube)-1||1.68||1000.00||130.00 |- | 40||TZHZ075 (H01-plate2)-1,2||||58.10||17.21||H9,H10||TZHZ075 (H01-plate2)-1||2.79||1000.00||130.00 |- | 41||PGP1iPS||||129.00||7.75||A11||PGP1iPS||12.25||1000.00||130.00 |- | 42||Hues1||||341.90||2.92||C11,C12||Hues1||17.08||1000.00||130.00 |- | 43||Hues3||||696.90||1.43||D11,D12||Hues3||18.57||1000.00||130.00 |- | 44||Hues8||||410.60||2.44||E11,E12||Hues8||17.56||1000.00||130.00 |} * Eluted volume 20ul with Elution buffer. After Qubit ss-DNA assay the total yields will be calculated based on ~17ul left in the well (or a little bit lower). == Qubit ssDNA assay == * Follow protocol provided with the kit * Took 1ul of bis-cvt DNA into 9ul of H2O, then added the diluted DNA to 190ul ssDNA Qubit reagent (dilution factor = 200) {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample ID''' | align="center" style="background:#f0f0f0;"|'''Well ID''' | align="center" style="background:#f0f0f0;"|'''Concentration in the Qubit (ng/mL)''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | align="center" style="background:#f0f0f0;"|'''Final conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Yields (ng)''' |- | 1. H9||A1||76.40||17.00||15.28||252.12 |- | **2. H9+Activin 100ng/mL 5days||B1||62.70||17.00||12.54||206.91 |- | 3. H9+BMP4 50ng/mL 5days||C1||110.00||17.00||22.00||363.00 |- | 4. HUViPS4F1||D1||117.00||17.00||23.40||386.10 |- | 5. HUViPS4F1+Activin 100ng/mL 5days||E1||76.90||17.00||15.38||253.77 |- | 6. HUViPS4F1+BMP4 50ng/mL 5days||F1||114.00||17.00||22.80||376.20 |- | 7. HUViPS4F3||G1||118.00||17.00||23.60||389.40 |- | 8. HUViPS4F3+Activin 100ng/mL 5days||H1||79.30||17.00||15.86||261.69 |- | 9. HUViPS4F3+BMP4 50ng/mL 5days||A3||98.90||17.00||19.78||326.37 |- | GM11995 (wellID G7)||B3||205.00||17.00||41.00||676.50 |- | GM11993 (wellID F2)||C3||80.80||17.00||16.16||266.64 |- | **GM10861 (wellID A9)||D3||51.80||17.00||10.36||170.94 |- | GM11992 (wellID C2 )||E3||112.00||17.00||22.40||369.60 |- | GM11994 (wellID G9 )||F3||138.00||17.00||27.60||455.40 |- | GM10860 (wellID F10)||G3||116.00||17.00||23.20||382.80 |- | GM12813 (wellID E8)||H3||176.00||17.00||35.20||580.80 |- | GM12802 (wellID E7)||A5||133.00||17.00||26.60||438.90 |- | GM12815 (wellID G12)||B5||140.00||17.00||28.00||462.00 |- | GM12812 (wellID F11)||C5||112.00||17.00||22.40||369.60 |- | GM12814 (wellID A7)||D5||97.10||17.00||19.42||320.43 |- | GM12801 (wellID D9)||E5||122.00||17.00||24.40||402.60 |- | NA12156 (wellID B3)-1||F5||72.20||17.00||14.44||238.26 |- | NA12156 (wellID B3)-2||G5||68.40||17.00||13.68||225.72 |- | NA12156 (wellID B3)-3||H5||76.30||17.00||15.26||251.79 |- | NA12156 (wellID B3)-4||A7||85.30||17.00||17.06||281.49 |- | NA12156 (wellID B3)-5||B7||81.00||17.00||16.20||267.30 |- | NA12156 (wellID B3)-6||C7||85.20||17.00||17.04||281.16 |- | NA12156 (wellID B3)-7||D7||65.70||17.00||13.14||216.81 |- | NA12156 (wellID B3)-8||E7||80.10||17.00||16.02||264.33 |- | NA12156 (wellID B3)-9||F7||69.30||17.00||13.86||228.69 |- | NA12156 (wellID B3)-10||G7||68.90||17.00||13.78||227.37 |- | **2HFF P6||H7||44.00||17.00||8.80||145.20 |- | **218 HFF P7||A9||41.20||17.00||8.24||135.96 |- | KEBR028A (B4-plate2)-1,2||B9||38.50||17.00||7.70||127.05 |- | ****KEBR028A (tube)-1,2||C9||Out Of Range||17.00||#VALUE!||#VALUE! |- | KEBR040 (D4-plate1)||D9||157.00||17.00||31.40||518.10 |- | TZHZ018 (F1-plate1)||E9||137.00||17.00||27.40||452.10 |- | TZHZ031A (tube)-1,2||F9||274.00||17.00||54.80||904.20 |- | TZHZ041A (tube)-1,2||G9||215.00||17.00||43.00||709.50 |- | TZHZ075 (H01-plate2)-1,2||H9||164.00||17.00||32.80||541.20 |- | **PGP1iPS||A11||30.00||17.00||6.00||99.00 |- | PGP1iPS-IMPRINT||B11||42.90||17.00||8.58||141.57 |- | **Hues1||C11||35.80||17.00||7.16||118.14 |- | **Hues3||D11||34.60||17.00||6.92||114.18 |- | **Hues8||E11||43.40||17.00||8.68||143.22 |} * Continued on 2011_09_18: [[http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2011-9-18]]
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